Controlled dissolution crosslinked protein crystals

ABSTRACT

The present invention relates to crosslinked protein crystals characterized by the ability to change from insoluble and stable form to soluble and active form upon a change in the environment of said crystals, said change being selected from the group consisting of change in temperature, change in pH, change in chemical composition, change from concentrate to dilute form, change in shear force acting upon the crystals and combinations thereof. According to one embodiment of this invention, such crosslinked protein crystals are capable of releasing their protein activity at a controlled rate. This invention also provides methods for producing such crosslinked protein crystals, methods using them for protein delivery and methods using them in cleaning agents, including detergents, pharmaceutical compositions, vaccines, personal care compositions, including cosmetics, veterinary compositions, foods, feeds, diagnostics and formulations for decontamination.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to crosslinked protein crystalscharacterized by the ability to change from insoluble and stable form tosoluble and active form upon a change in the environment surroundingsaid crystals, said change being selected from the group consisting ofchange in temperature, change in pH, change in chemical composition,change from concentrate to dilute form, change in shear force actingupon the crystals and combinations thereof. According to one embodimentof this invention, such crosslinked protein crystals are capable ofreleasing their protein activity at a controlled rate. This inventionalso provides methods for producing such crosslinked protein crystals,methods using them for protein delivery and methods using them incleaning agents, including detergents, pharmaceutical compositions,vaccines, personal care compositions, including cosmetics, veterinarycompositions, foods, feeds, diagnostics and formulations fordecontamination.

BACKGROUND OF THE INVENTION

[0002] Proteins are used in a wide range of applications in the fieldsof industrial chemistry, pharmaceuticals, veterinary products, cosmeticsand other consumer products, foods, feeds, diagnostics anddecontamination. At times, such uses have been limited by constraintsinherent in proteins themselves or imposed by the environment or mediain which they are used. Such constraints may result in poor stability ofthe proteins, variability of performance or high cost. In order forproteins to realize their full potential in the fields in which they areused, they must be able to function without excessive intervention bytheir surrounding environment. In the past, environmental elements haveoften posed barriers to the widespread use of proteins.

[0003] Various approaches have been employed to overcome these barriers.However, these approaches have incurred either loss of protein activityor the additional expense of protein stabilizing carriers orformulations.

[0004] One unique approach to overcoming barriers to the widespread useof proteins is crosslinked enzyme crystal (“CLEC™) technology [N. L. St.Clair and M. A., Navia, J. Am. Chem. Soc., 11.4, pp. 4314-16 (1992)].Crosslinked enzyme crystals retain their activity in environments thatare normally incompatible with enzyme function. Such environmentsinclude prolonged exposure to proteases and other protein digestionagents, high temperature or extreme pH. In such environments,crosslinked enzyme crystals remain insoluble and stable.

[0005] Protein solubility, leading to controlled release or dissolutionof protein, is important in many industrial fields. Such industriesinclude those concerning cleaning agents, including detergents,pharmaceuticals, consumer and personal care products, veterinaryproducts, foods, feeds, diagnostics and decontamination. Variousapproaches to controlled release have been proposed. These includeencapsulation, such as that described in U.S. Pat. Nos. 4,579,779 and5,500,223. Other approaches include the use of mechanical or electricalfeed devices and osmotic pumps.

[0006] Controlled release in the pharmaceutical field has been addressedby various means. U.S. Pat. No. 5,569,467 refers to the use of sustainedrelease microparticles comprising a biocompatible polymer and apharmaceutical agent, which is released as the polymer degrades. U.S.Pat. No. 5,603,956 refers to solid, slow release pharmaceutical dosageunits comprising crosslinked amylase, alpha amylase and a pharmaceuticalagent. U.S. Pat. No. 4,606,909 refers to oral, controlled-releasemultiple unit formulations in which homogeneous cores containingparticles of sparingly soluble active ingredients are coated with apH-sensitive erodable coating. U.S. Pat. No. 5,593,697 refers topharmaceutical or veterinary implants comprising a biologically activematerial, an excipient comprising at least one water soluble materialand at least one water insoluble material and a polymer film coatingadapted to rupture at a predetermined period of time after implant.

[0007] The objective of controlled release of proteins, however, must bebalanced with the fact that the protein itself may not be stable understorage conditions. Protein stability may also be adversely affected byother components of the formulation in which it is contained. Forexample, heavy duty liquid detergents constitute hostile environmentsfor component enzymes. Such problems have been approached through theuse of mutant subtilisin proteases, which are said to have improvedoxidative stability. See U.S. Pat. No. 4,760,025 and PCT patentapplication WO89/06279. Proteins, the enzymes most widely used indetergents, catalyze their own decomposition. Strategies such as theaddition of protease inhibitors (e.g., borate with glycols) or thelowering of water activity have been only partially effective.

[0008] Another approach, described in U.S. Pat. No. 5,385,959, isencapsulation of degradation-sensitive detergent components in capsulesof composite emulsion polymers, which permit dilution release thereof.U.S. Pat. No. 5,286,404 refers to a liquid detergent composition said tohave improved enzyme solubility while preserving enzyme activity. Theimprovement is attributed to chemical modification of free primary aminogroups in an enzyme solution via aldehyde treatment, acylation oralkylation.

[0009] Despite such progress in protein technology generally, the needstill exists for proteins which are stable under conditions of storage,while active under conditions of use.

DISCLOSURE OF THE INVENTION

[0010] The present invention relates to crosslinked protein crystalscharacterized by the ability to change from insoluble and stable form tosoluble and active form upon a change in the environment surroundingsaid crystals, said change being selected from the group consisting ofchange in temperature, change in pH, change in chemical composition,change from concentrate to dilute form, change in shear force actingupon the crystals and combinations thereof. According to one embodimentof this invention, such crosslinked protein crystals are capable ofreleasing their protein activity at a controlled rate.

[0011] Advantageously, the crosslinked protein crystals of thisinvention are insoluble and stable under storage conditions and solubleand active under conditions of use.

[0012] This invention also provides cleaning agents, includingdetergents, pharmaceutical compositions, vaccines, personal carecompositions, including cosmetics, veterinary compositions, foods,feeds, diagnostics and formulations for decontamination. Additionally,this invention includes methods for producing such crosslinked proteincrystals and methods for protein delivery using them.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a graph representing the stability of various enzymes inCiba detergent # 16 at 40° C.

[0014]FIG. 2 is a graph representing the washing performance of liquiddetergent formulations, including a formulation containing crosslinkedsubtilisin crystals according to the present invention, on fabric soiledwith blood, milk and carbon black.

[0015]FIG. 3 is a graph representing the washing performance of liquiddetergent formulations, including a formulation containing crosslinkedsubtilisin crystals according to the present invention, after storage at30° C., on fabric soiled with cocoa.

[0016]FIG. 4 is a graph representing the washing performance of liquiddetergent formulations, including a formulation containing crosslinkedsubtilisin crystals according to the present invention, after storage at40° C., on fabric soiled with cocoa.

[0017]FIG. 5 is a graph representing the washing performance of liquiddetergent formulations, including a formulation containing crosslinkedsubtilisin crystals according to the present invention, after storage at30° C., on fabric soiled with blood, milk and carbon black.

[0018]FIG. 6 is a graph representing the washing performance of liquiddetergent formulations, including a formulation containing crosslinkedsubtilisin crystals according to the present invention, after storage at40° C., on fabric soiled with blood, milk and carbon black.

[0019]FIG. 7 is a graph representing the washing performance of liquiddetergent formulations, including a formulation containing crosslinkedsubtilisin crystals according to the present invention, after storage at30° C., on fabric soiled with blood.

[0020]FIG. 8 is a graph representing the solubility of crosslinkedsubtilisin crystals according to the present invention at 30° C.

[0021]FIG. 9 is a graph representing the solubility of crosslinkedsubtilisin crystals according to the present invention at 37° C.

DETAILED DESCRIPTION OF THE INVENTION

[0022] In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth. In thedescription, the following terms or phrases are employed:

[0023] Aqueous-organic solvent mixture—a mixture comprising n % organicsolvent, where n is between 1 and 99 and m % aqueous, where m is 100-n.

[0024] Catalytically effective amount—an amount of crosslinked proteincrystals of this invention which is effective to treat, protect, repairor detoxify the area to which they are applied over some period of time.

[0025] Change in chemical composition—any change in the chemicalcomponents of the environment surrounding the crosslinked proteincrystals that affects the environment or the crosslinker, includingaddition of chemical reagents, chemical changes induced by applicationof energy in the form of light, microwave, or radiation to theenvironment, chemical events that affect the crosslinker andcombinations thereof.

[0026] Change in shear force acting upon the crystals—any change infactors of the environment surrounding the crosslinked protein crystalsunder conditions of use, such as, changes in mechanical pressure, bothpositive and negative, revolution stirring, centrifugation, tumbling,mechanical agitation and filtration pumping.

[0027] Controlled dissolution—dissolution of crosslinked proteincrystals or release of the protein constituent of said crystals that is(1) triggered by a change in the environment surrounding said crystals,said change being selected from the group consisting of change intemperature, change in pH, change in chemical composition, change fromconcentrate to dilute form, change in shear force acting upon thecrystals and combinations thereof and (2) controlled by a factorselected from the group consisting of the degree of crosslinking of saidcrosslinked protein crystals, the length of time of exposure of proteincrystals to the crosslinking agent, the rate of addition of crosslinkingagent to said protein crystals, the nature of the crosslinker, the chainlength of the crosslinker, the surface area of said crosslinked proteincrystals, the size of said crosslinked protein crystals, the shape ofsaid crosslinked protein crystals and combinations thereof. As usedherein, the phrase “controlled dissolution” does not include leaching.

[0028] Formulations for decontamination—formulations selected from thegroup consisting of: formulations for decontamination of chemicalwastes, herbicides, insecticides, pesticides, environmental hazards andchemical warfare agents.

[0029] Insoluble and stable form of a protein—a form of a protein whichis insoluble in aqueous solvents, organic solvents or aqueous-organicsolvent mixtures and which displays greater stability than the solubleform of the protein. According to an alternate embodiment of thisinvention, the phrase “insoluble and stable form of a protein” may be aprotein which is insoluble in dry formulations but soluble in wetformulations. In any embodiment, the crosslinked protein crystals may beactive in insoluble form. And in one embodiment, the crosslinked proteincrystals may be active in insoluble form, then dissolve or are removedor digested once their function is complete.

[0030] Organic solvents—any solvent of non-aqueous origin.

[0031] Pharmaceutically effective amount—an amount of crosslinkedprotein crystals which is effective to treat a condition in anindividual to whom they are administered over some period of time.

[0032] Prophylactically effective amount—an amount of crosslinkedprotein crystals which is effective to prevent a condition in anindividual to whom they are administered over some period of time.

[0033] Protein—any peptide having a tertiary structure or any protein.

[0034] Protein activity—an activity selected from the group consistingof binding, catalysis, activities which generate a functional responsewithin the environment in which the protein used, such as induction ofimmune response and immunogenicity, or combinations thereof.

[0035] Protein activity release rate—the quantity of protein dissolvedper unit time.

[0036] The crosslinked protein crystals of this invention areparticularly advantageous because they are stable in harsh environmentsimposed by the formulations or compositions in which they are employedor conditions of their storage. At the same time, these crosslinkedprotein crystals are capable of (1) change to soluble and active form(an active form including, in one embodiment of this invention, a formwhich is active against macromolecular substrates) or

[0037] (2) controlled dissolution or release of their activity whenexposed to one or more triggers in their environment. Such triggers maybe selected from the group consisting of change in temperature, changein pH, change in chemical composition, change from concentrate to diluteform, change in shear force acting upon the crystals and combinationsthereof. Controlled dissolution or release of activity of crosslinkedprotein crystals according to this invention may also be triggered overa change in time.

[0038] Specific examples of such triggers include an increase ordecrease in temperature, for example, an increase in temperature from alow temperature between about 0° C. and about 20° C. to a hightemperature between about 25° C. and about 70° C. Other triggers includea change from acidic pH to basic pH and a change from basic pH to acidicpH. Examples of triggers of change from concentrate to dilute forminclude, for example, a change in solute concentration, a change inconcentration of all solutes from about 2-fold to about 10,000-fold, achange in concentration of all solutes from about 2-fold to about700-fold, an increase or decrease in salt concentration, an increase ordecrease in water concentration, an increase or decrease in organicsolvent concentration, a decrease in protein concentration and adecrease in detergent concentration.

[0039] Additional triggers involve changes in chemical composition ofthe environment surrounding the crosslinked protein crystals that affectthe environment or the crosslinker itself. Such changes include, forexample, addition of chemical reagents, increase or decrease in organicsolvent concentration, chemical events that affect the crosslinker,chemical changes induced by application of energy, including light,microwave or radiation. As explained above, any of these triggers mayact in combination or in sequence with one or more of the othertriggers.

[0040] Controlled dissolution of crosslinked protein crystals accordingto the present invention may also be effected by a change in timesufficient to permit a protein activity release rate between about 0.1%per day and about 100% per day, a change in time sufficient to permit aprotein activity release rate between about 0.01% per hour and about100% per hour and a change in time sufficient to permit a proteinactivity release rate between about 1% per minute and about 50% perminute.

[0041] Crosslinked protein crystals according to this invention,therefore, include those capable of releasing their protein activity ata controlled rate upon exposure to a change in their environment, saidchange being selected from the group consisting of change in pH, changein solute concentration, change in temperature, change in chemicalcomposition, change in shear force acting upon the crystals andcombinations thereof. Said controlled rate of releasing protein activitymay be determined by a factor selected from the group consisting of thedegree of crosslinking of the crosslinked protein crystals, the lengthof time of exposure of protein crystals to the crosslinking agent, therate of addition of crosslinking agent to the protein crystals, thenature of the crosslinker, the chain length of the crosslinker, thesurface area of the crosslinked protein crystals, the size of thecrosslinked protein crystals, the shape of the crosslinked proteincrystals and combinations thereof.

[0042] As a result of their crystalline nature, the crosslinked proteincrystals of this invention achieve uniformity across the entirecrosslinked crystal volume. This uniformity is maintained by theintermolecular contacts and chemical crosslinks between the proteinmolecules constituting the crystal lattice. The protein moleculesmaintain a uniform distance from each other, forming well-defined stablepores within the crosslinked protein crystals that facilitate access ofsubstrate to the protein, as well as removal of product. In thesecrosslinked protein crystals, the lattice interactions, when fixed bychemical crosslinks, are particularly important in providing stabilityand preventing denaturation, especially in storage, under conditionsincluding harsh environments created by components of compositions inwhich the crystals are used. At the same time, the protein crystals arecrosslinked in such a way that they dissolve or release their proteinactivity upon exposure to a trigger in their environment encounteredunder conditions of use. Thus, they may be substantially insoluble andstable in a composition under storage conditions and substantiallysoluble and active under conditions of use of said composition.

[0043] Factors contributing to the release rate of protein activity ofcrosslinked protein crystals according to this invention include thedegree of crosslinking of the crosslinked protein crystals, the lengthof time of exposure of protein crystals to the crosslinking agent, therate of addition of crosslinking agent to the protein crystals, thelength of time of exposure of protein crystals to the crosslinkingagent, the nature of the crosslinker, the chain length of thecrosslinker, the surface area of the crosslinked protein crystals, thesize of the crosslinked protein crystals, the shape of the crosslinkedprotein crystals and combinations thereof.

[0044] In addition to their activity, crosslinked protein crystalsaccording to this invention are particularly stable and insoluble understorage conditions, including the attendant storage temperature, storagepH, storage time, storage concentrate form, storage involving little orno shear force acting upon the crystals, or combinations thereof.Advantageously, these crosslinked protein crystals are soluble andactive under conditions of use, including conditions involving change intemperature, change in pH, change in chemical composition, change fromconcentrate to dilute form, change in shear force acting upon thecrystals and combinations thereof. Such properties make the crosslinkedprotein crystals of this invention particularly useful for delivery ofcleaning agents, including detergents, pharmaceuticals, personal careagents or compositions, including cosmetics, vaccines, veterinarycompositions, foods, feeds, diagnostics and formulations fordecontamination.

[0045] According to one embodiment, the crosslinked protein crystals ofthis invention are characterized by a half-life of activity understorage conditions which is greater than at least 2 times that of thesoluble form of the protein that is crystallized to form the crystalsthat are crosslinked and activity similar to that of the soluble form ofthe protein under conditions of use.

[0046] The protein constituent of the crosslinked protein crystals ofthis invention may be any protein, including, for example, therapeuticproteins, prophylactic proteins, including antibodies, cleaning agentproteins, including detergent proteins, personal care proteins,including cosmetic proteins, veterinary proteins, food proteins, feedproteins, diagnostic proteins and decontamination proteins. Includedamong such proteins are enzymes, such as, for example, hydrolases,isomerases, lyases, ligases, transferases and oxidoreductases. Examplesof hydrolases include thermolysin, elastase, esterase, lipase,nitrilase, amylase, pectinase, subtilinase, hydantoinase, asparaginase,urease, subtilisin and other proteases and lysozyme. Examples of lyasesinclude aldolases and hydroxynitrile lyase. Examples of oxidoreductasesinclude peroxidase, laccase, glucose oxidase, alcohol dehydrogenase andother dehydrogenases. Other enzymes which may be crystallized andcrosslinked include cellulases and oxidases.

[0047] Examples of therapeutic or prophylactic proteins includehormones, antibodies, inhibitors, growth factors, trophic factors,cytokines, lymphokines, toxoids, erythropoietin, Factor VIII, insulin,amylin, TPA, dornase-α, α-1-antitripsin, human growth hormones, nervegrowth hormones, bone morphogenic proteins, urease, toxoids, fertilityhormones, FSH, LSH, postridical hormones, tetanus toxoid, diptheriatoxoid, vitamins and nutrients.

[0048] According to one embodiment of this invention, crosslinkedprotein crystals are characterized by activity which is similar to thatof their soluble or uncrosslinked crystallized counterparts underconditions of use. Advantageously however, the crosslinked proteincrystals of this invention display improved stability under storageconditions, as compared to their soluble or uncrosslinked crystallizedcounterpart proteins.

[0049] The crosslinked protein crystals of this invention may be used inany of a number of chemical processes. Such processes include industrialand research-scale processes, such as organic synthesis of specialtychemicals and pharmaceuticals. Enzymatic conversion processes includeoxidations, reductions, additions, including esterifications andtransesterifications, hydrolyses, eliminations, rearrangements, andasymmetric conversions, including stereoselective, stereospecific andregioselective reactions.

[0050] Thus, crosslinked protein crystals according to this inventionmay be advantageously used instead of conventional soluble orimmobilized proteins in cleaning agents, including detergents,pharmaceuticals, veterinary compounds, personal care compositions,including cosmetics, foods, feeds, vaccines, pulp, paper and textileprocessing, diagnostics and formulations for decontamination.

[0051] Crosslinked protein crystals according to this invention may alsobe used in various environmental applications. They may be used in placeof conventional soluble or immobilized proteins for environmentalpurposes, such wide area decontamination of environmental hazards.

[0052] Alternatively, the crosslinked protein crystals of this inventionmay be used in cleaning agents, selected from the group consisting ofdetergents, such as powdered detergents and liquid detergents, bleaches,household cleaners, hard surface cleaners, industrial cleaners andcarpet and upholstery shampoos.

[0053] Cleaning agents containing crosslinked protein crystals accordingto the present invention may also comprise compounds conventionallyincluded in such agents. See, for example, Soaps and Detergents. ATheoretical and Practical Review, Louis Spitz (Ed.), AOCS Press(Champlain, Ill.) (1996). Such compounds include anionic, non-ionic,cationic or zwitterionic surfactants, or mixtures thereof.

[0054] Anionic surfactants are exemplified by alkyl sulfates, alkylether sulfates, alkyl sulfonates, alkylaryl sulfonates, olefinsulfonates, alkyl ether phosphates, alkyl ether phosphates, fatty acidsalts, soaps, isothionates and sulfonated unsaturated esters and acids.

[0055] Non-ionic surfactants are exemplified by products of condensationof an organic aliphatic or alkyl aromatic hydrophobic compound with analkylene oxide, alkyl polyglucosides and sugar esters.

[0056] Cationic surfactants are exemplified by quarternary ammoniumsalts of tertiary alkyl amines, amino amides, amino esters orimidazolines containing al least one long chain (C₈-C₂₂) aliphatic groupor an alkyl-aryl group, wherein alkyl comprises about 4 to 12 carbonatoms and aryl is preferably a phenylene group.

[0057] Zwitterionic surfactants are exemplified by derivatives ofquarternary ammonium, quarternary phosphonium or tertiary sulfoniumcompounds, derivatives of secondary and tertiary amines and derivativesof heterocyclic secondary and tertiary amines.

[0058] And crosslinked protein crystals according to this invention maybe used as ingredients in personal care compositions, includingcosmetics, such as creams, lotions, emulsions, foams, washes, compacts,gels, mousses, slurries, powders, sprays, pastes, ointments, salves,balms, drops, shampoos, and sunscreens. In topical creams and lotions,for example, they may be used as humectants or for skin protection,softening, bleaching, cleaning, deproteinization, lipid removal,moisturizing, decoloration, coloration or detoxification. They may alsobe used as anti-oxidants in cosmetics.

[0059] According to this invention, any individual, including humans andother mammals, may be treated in a pharmaceutically acceptable mannerwith a pharmaceutically effective or a catalytically effective amount ofcrosslinked protein crystals for a period of time sufficient to treat acondition in the individual to whom they are administered over someperiod of time. Alternatively, individuals may receive aprophylactically effective or a catalytically effective amount ofcrosslinked protein crystals of this invention which is effective toprevent a condition in the individual to whom they are administered oversome period of time.

[0060] Such crosslinked protein crystals may be administered alone, aspart of a pharmaceutical, personal care or veterinary preparation or aspart of a prophylactic preparation, such as a vaccine, with or withoutadjuvant. They may be administered by parenteral or non-parenteralroute. For example, they may be administered by oral, pulmonary, nasal,aural, anal, dermal, ocular, intravenous, intramuscular, intraarterial,intraperitoneal, mucosal, sublingual, subcutaneous, or intracranialroute. In either pharmaceutical, personal care or veterinaryapplications, crosslinked protein crystals may be topically administeredto any epithelial surface. Such epithelial surfaces include oral,ocular, aural, anal and nasal surfaces, to treat, protect, repair ordetoxify the area to which they are applied.

[0061] The present invention also includes controlled releaseformulations comprising crosslinked protein crystals according to thisinvention. In such formulations, the crosslinked protein crystals aresubstantially insoluble under storage conditions and capable ofreleasing their protein activity in vivo at a controlled rate. Forexample, a pharmaceutical controlled release formulation according tothis invention, administered by oral route, is characterized in that thecomponent crosslinked protein crystals are substantially insoluble undergastric pH conditions and substantially soluble under small intestine pHconditions. Alternatively, for these and other uses according to thisinvention, the crosslinked protein crystals may be active in theinsoluble form and then dissolve and are removed or digested once theirfunction is complete.

[0062] Pharmaceutical, personal care, veterinary or prophylacticcompositions comprising crosslinked protein crystals according to thisinvention may also be selected from the group consisting of tablets,liposomes, granules, spheres, microparticles, microspheres and capsules.

[0063] For such uses, as well as other uses according to this invention,crosslinked protein crystals may be formulated into tablets. Suchtablets constitute a liquid-free, dust-free form of crosslinked proteincrystal storage which are easily handled and retain acceptable levels ofactivity.

[0064] Alternatively, the crosslinked protein crystals may be in avariety of conventional depot forms employed for administration toprovide reactive compositions. These include, for example, solid,semi-solid and liquid dosage forms, such as liquid solutions orsuspensions, gels, creams, balms, emulsions, lotions, slurries, powders,sprays, foams, pastes, ointments, salves, balms and drops.

[0065] Compositions or formulations comprising the crosslinked proteincrystals of this invention may also comprise any conventional carrier oradjuvant used in pharmaceuticals, personal care compositions orveterinary formulations. These carriers and adjuvants include, forexample, Freund's adjuvant, ion exchangers, alumina, aluminum stearate,lecithin, buffer substances, such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium, trisilicate, cellulose-based substances andpolyethylene glycol. Adjuvants for topical or gel base forms mayinclude, for example, sodium carboxymethylcellulose, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol andwood wax alcohols.

[0066] According to one embodiment of this invention, crosslinkedprotein crystals may be combined with any conventional materials usedfor controlled release administration. Such materials include, forexample, coatings, shells and films, such as enteric coatings andpolymer coatings and films.

[0067] The most effective mode of administration and dosage regimen offormulations or compositions comprising crosslinked protein crystals ofthis invention will depend on the effect desired, previous therapy, ifany, the individual's health status or status of the condition itselfand response to the crosslinked protein crystals and the judgment of thetreating physician or clinician. The crosslinked protein crystals may beadministered in any dosage form acceptable for pharmaceuticals, personalcare compositions or veterinary formulations, at one time or over aseries of treatments.

[0068] The amount of the crosslinked protein crystals that may becombined with carrier materials to produce a single dosage form willvary depending upon the particular mode of administration, formulation,dose level or dose frequency. A typical preparation will contain betweenabout 0.01% and about 99%, preferably between about 1% and about 50%,crosslinked protein crystals (w/w).

[0069] Upon improvement of the individual's condition, a maintenancedose of crosslinked protein crystals may be administered, if necessary.Subsequently, the dosage or frequency of administration, or both, may bereduced as a function of the symptoms, to a level at which the improvedcondition is retained. When the condition has been alleviated to thedesired level, treatment should cease. Individuals may, however, requireintermittent treatment on a long-term basis upon any recurrence of thecondition or symptoms thereof.

[0070] An alternate embodiment of the present invention includes proteindelivery systems comprising the crosslinked protein crystals disclosedherein. Such a system may be used to deliver proteins such as thoseincluded in cleaning agents, such as detergents, personal care products,such as cosmetics, pharmaceuticals, veterinary compositions, vaccines,foods, feeds, diagnostics and formulations for decontamination. Proteindelivery systems of this invention, which may be formulations ordevices, such as implantable devices, may be microparticulate proteindelivery systems, wherein the crosslinked protein crystals have alongest dimension between about 0.01 μm and about 500 μm, alternativelybetween about 0.1 μm and about 50 μm. The crosslinked protein crystalcomponents of such systems may have a shape selected from the groupconsisting of: spheres, needles, rods, plates, such as hexagons andsquares, rhomboids, cubes, bipryamids and prisms. Advantageously, thecrosslinked crystal form of the proteins of this invention allow loadingof up to between about 50% and about 90% protein per unit of weight.

[0071] One example of a protected protein system according to thisinvention is suitable for storage in a medium such as a liquiddetergent, prior to use. The crosslinked protein crystal components ofsuch a system are insoluble under storage conditions in saidmedium—which typically causes degradation of the soluble form of theprotein that is crystallized to form said crystal that iscrosslinked—and soluble under conditions of use.

[0072] According to the present invention, preparation of crosslinkedprotein crystals includes the steps of crystallizing and crosslinkingthe protein. This may be carried out as illustrated below.

[0073] Preparation of Crosslinked Protein Crystals—ProteinCrystallization

[0074] Protein crystals are grown by controlled crystallization ofprotein out of aqueous solution or aqueous solution-containing organicsolvents. Conditions to be controlled include, for example, the rate ofevaporation of solvent, the presence of appropriate co-solutes andbuffers, pH and temperature. A comprehensive review of the variousfactors affecting the crystallization of proteins has been published byMcPherson, Methods Enzymol., 114, pp. 112-20 (1985).

[0075] McPherson and Gilliland, J. Crystal Growth, 90, pp. 51-59 (1988)have compiled comprehensive lists of proteins and nucleic acids thathave been crystallized, as well as the conditions under which they werecrystallized. A compendium of crystals and crystallization recipes, aswell as a repository of coordinates of solved protein and nucleic acidstructures, is maintained by the Protein Data Bank at the BrookhavenNational Laboratory [http//www.pdb.bnl.gov; Bernstein et al., J. Mol.Biol., 112, pp. 535-42 (1977)]. These references can be used todetermine the conditions necessary for crystallization of a protein, asa prelude to the formation of an appropriate crosslinked proteincrystal, and can guide the crystallization strategy for other proteins.Alternatively, an intelligent trial and error search strategy can, inmost instances, produce suitable crystallization conditions for manyproteins, provided that an acceptable level of purity can be achievedfor them [see, e.g., C. W. Carter, Jr. and C. W. Carter, J. Biol. Chem.,254, pp. 12219-23 (1979)].

[0076] For use in crosslinked protein crystals according to thisinvention, the large single crystals which are needed for X-raydiffraction analysis are not required. Microcrystalline showers aresuitable.

[0077] For example, the crosslinked protein crystals may have a longestdimension between about 0.01 μm and about 500 μm, alternatively, between0.1 μm and about 50 μm. They may also have a shape selected from thegroup consisting of: spheres, needles, rods, plates, such as hexagonsand squares, rhomboids, cubes, bipryamids and prisms.

[0078] In general, crystals are produced by combining the protein to becrystallized with an appropriate aqueous solvent or aqueous solventcontaining appropriate crystallization agents, such as salts or organicsolvents. The solvent is combined with the protein and subjected toagitation at a temperature determined experimentally to be appropriatefor the induction of crystallization and acceptable for the maintenanceof protein activity and stability. The solvent can optionally includeco-solutes, such as divalent cations, co-factors or chaotropes, as wellas buffer species to control pH. The need for co-solutes and theirconcentrations are determined experimentally to facilitatecrystallization. In an industrial-scale process, the controlledprecipitation leading to crystallization can best be carried out by thesimple combination of protein, precipitant, co-solutes and, optionally,buffers in a batch process. Alternative laboratory crystallizationmethods, such as dialysis or vapor diffusion, can also be adopted.McPherson, supra and Gilliland, supra, include a comprehensive list ofsuitable conditions in their reviews of the crystallization literature.Occasionally, incompatibility between the crosslinking agent and thecrystallization medium might require exchanging the crystals into a moresuitable solvent system.

[0079] Many of the proteins for which crystallization conditions havealready been described, may be used to prepare crosslinked proteincrystals according to this invention. It should be noted, however, thatthe conditions reported in most of the above-cited references have beenoptimized to yield, in most instances, a few large, diffraction qualitycrystals. Accordingly, it will be appreciated by those of skill in theart that some degree of adjustment of these conditions to provide a highyielding process for the large scale production of the smaller crystalsused in making crosslinked protein crystals may be necessary.

[0080] Preparation of Crosslinked Protein Crystals—Crosslinking ofProtein Crystals

[0081] Once protein crystals have been grown in a suitable medium theycan be crosslinked. Crosslinking results in stabilization of the crystallattice by introducing covalent links between the constituent proteinmolecules of the crystal. This makes possible transfer of the proteininto an alternate environment that might otherwise be incompatible withthe existence of the crystal lattice or even with the existence ofintact protein.

[0082] Advantageously, crosslinking according to the present inventionis carried out in such a way that, under conditions of storage, thecrosslinking interactions prevent the constituent protein molecules inthe crystal from going back into solution, effectively insolubilizing orimmobilizing the protein molecules into microcrystalline particles. Uponexposure to a trigger in the environment surrounding the crosslinkedprotein crystals, such as under conditions of use rather than storage,the protein molecules dissolve, releasing their protein activity. Therate of dissolution is controlled by one or more of the followingfactors: the degree of crosslinking, the length of time of exposure ofprotein crystals to the crosslinking agent, the rate of addition ofcrosslinking agent to the protein crystals, the nature of thecrosslinker, the chain length of the crosslinker, the surface area ofthe crosslinked protein crystals, the size of the crosslinked proteincrystals, the shape of the crosslinked protein crystals and combinationsthereof.

[0083] Crosslinking can be achieved using one or a combination of a widevariety of multifunctional reagents, at the same time (in parallel) orin sequence, including bifunctional reagents. Upon exposure to a triggerin the surrounding environment, or over a given period of time, thecrosslinks between protein crystals crosslinked with suchmultifunctional crosslinking agents lessen or weaken, leading to proteindissolution or release of activity. Alternatively, the crosslinks maybreak at the point of attachment, leading to protein dissolution orrelease of activity. Such crosslinking agents include glutaraldehyde,succinaldehyde, octanedialdehyde and glyoxal. Additional multifunctionalcrosslinking agents include halo-triazines, e.g., cyanuric chloride;halo-pyrimidines, e.g., 2,4,6-trichloro/bromo-pyrimidine; anhydrides orhalides of aliphatic or aromatic mono- or di-carboxylic acids, e.g.,maleic anhydride, (meth)acryloyl chloride, chloroacetyl chloride;N-methylol compounds, e.g., N-methylol-chloro acetamide; di-isocyanatesor di-isothiocyanates, e.g., phenylene-1,4-di-isocyanate and aziridines.Other crosslinking agents include epoxides, such as, for example,di-epoxides, tri-epoxides and tetra-epoxides. According to a preferredembodiment of this invention, the crosslinking agent is glutaraldehyde,used alone or in sequence with an epoxide. For a representative listingof other available crosslinking reagents see, for example, the 1996catalog of the Pierce Chemical Company. Such multifunctionalcrosslinking agents may also be used, at the same time (in parallel) orin sequence, with reversible crosslinking agents, such as thosedescribed below.

[0084] According to an alternate embodiment of this invention,crosslinking may be carried out using reversible crosslinkers, inparallel or in sequence. The resulting crosslinked protein crystals arecharacterized by a reactive multi-functional linker, into which atrigger is incorporated as a separate group. The reactive functionalityis involved in linking together reactive amino acid side chains in aprotein and the trigger consists of a bond that can be broken byaltering one or more conditions in the surrounding environment (e.g.,pH, temperature, or thermodynamic water activity). This is illustrateddiagrammatically as:

X-Y-Z+2 AA residues-->AA₁-X-Y-Z-AA₂

change in environment-->AA₁-X+Y-Z-AA₂

[0085] where X and Z are groups with reactive functionality

[0086] where Y is a trigger

[0087] where AA₁ and AA₂ represent reactive amino acid residues on thesame protein or on two different proteins. The bond between thecrosslinking agent and the protein may be a covalent or ionic bond, or ahydrogen bond. The change in surrounding environment results in breakingof the trigger bond and dissolution of the protein. Thus, the crosslinksbetween protein crystals crosslinked with such reversible crosslinkingagents break, leading to protein crystal dissolution or release ofactivity.

[0088] Alternatively, the reactive functionality of the crosslinker andthe trigger may be the same, as in:

X-Z+2AA residues-->AA₁-X-Z-AA₂

change in environment-->AA₁+X-Z-AA₂.

[0089] The crosslinker may be homofunctional (X═Y) or heterofunctional(X is not equal to Y). The reactive functionality X and Y may be, butnot limited to the following functional groups (where R, R′, R″, and R′″may be alkyl, aryl or hydrogen groups):

[0090] I. Reactive acyl donors are exemplified by: carboxylate estersRCOOR′, amides RCONHR′, Acyl azides RCON₃, carbodiimides R—N═C═N—R′,N-hydroxyimide esters, RCO—O—NR′, imidoesters R—C═NH2⁺(OR′), anhydridesRCO—O—COR′, carbonates RO—CO—O—R′, urethanes RNHCONHR′, acid halidesRCOHal (where Hal=a halogen), acyl hydrazides RCONNR′R″, O-acylisoureasRCO—O—C═NR′(—NR″R′″),

[0091] II. Reactive carbonyl groups are exemplified by: aldehydes RCHOand ketones RCOR′, acetals RCO(H₂)R′, ketals RR′CO₂R′R″. Reactivecarbonyl containing functional groups known to those well skilled in theart of protein immobilization and crosslinking are described in theliterature [Pierce Catalog and Handbook, Pierce Chemical Company,Rockford, Ill. (1994); S. S. Wong, Chemistry of Protein Conjugation andCross-Linking, CRC Press, Boca Raton, Fla. (1991)].

[0092] III. Alkyl or aryl donors are exemplified by: alkyl or arylhalides R-Hal, azides R—N₃, sulfate esters RSO₃R′, phosphate estersRPO(OR′₃), alkyloxonium salts R₃O+, sulfonium R₃S+, nitrate estersRONO₂, Michael acceptors RCR′═CR′″COR″, aryl fluorides ArF, isonitrilesRN+≡C—, haloamines R₂N-Hal, alkenes and alkynes.

[0093] IV. Sulfur containing groups are exemplified by disulfides RSSR′,sulfhydryls RSH, epoxides R₂C ^(O) CR′₂.

[0094] V. Salts are exemplified by alkyl or aryl ammonium salts R₄N+,carboxylate RCOO—, sulfate ROSO₃—, phosphate ROPO₃″ and amines R₃N.

[0095] The table below includes examples of triggers, organized byrelease mechanism. In the table, R═ is a multifunctional crosslinkingagent that can be an alkyl, aryl, or other chains with activating groupsthat can react with the protein to be crosslinked. Those reactive groupscan be any variety of groups such as those susceptible to nucleophilic,free radical or electrophilic displacement including halides, aldehydes,carbonates, urethanes, xanthanes, epoxides among others. Release TriggerExamples Conditions 1. Acid Labile R—O—R H⁺ or Lewis Linkers e.g. Thp,MOM, Acidic catalysts Acetal, ketal Aldol, Michael adducts, esters 2.Base Labile R′OCO2—R′ Variety of basic Linkers Carbonates mediaR′O—CONR₂ Carbamates R₂′NCONR₂ Urethanes Aldol, Michael adducts, esters3. Fluoride R—OSiR₃ Aqueous F⁻ Labile Linkers Various Si containinglinkers 4. Enzyme RCOOR, RCONR₂′ Free lipases, Labile Linkers amidases,esterases 5. Reduction Disulfide H₂ catalyst; Labile Linkers linkersthat Hydrides cleave via Hydrogenolysis Reductive Elimination R′—S—S—R6. Oxidation R—OSiR₃ Oxidizing Labile Linkers Glycols R— agents: e.g.CH(OH)—CH(OH)—R′ H₂O₂, NaOCl, IO₄ ⁻ Metal based oxidizers, otherhypervalent oxidents 7. Thio-labile R′—S—S—R Thiols, e.g., linkers Cys,DTT, mercaptoethanol 8. Heavy Metal Various Allyl Transition metalLabile Linkers Ethers based reagents ROCH₂CH═CHR (Pd, Ir, Hg, Ag, Alkyl,Acyl Cu, Tl, Rh) Allyl ester Pd(0) catalysts 9. PhotolabileO-nitrobenzyl light (hv) Linkers (ONB) DESYL groups in linker 10. FreeThiohydroxamate Free radical Radical Labile ester initiator Linkers(Barton ester) 11. Metal- Iron (III) Metal removal chelate linkeddiphenanthroline e.g. by chelation or precipitation 12. ThermallyPeroxides Increase in Labile Linkers R—OO—R temperature 13. “SafetyMethylthioethyl Base; amines, Catch” Labile (Mte) others LinkersDithianes

[0096] Additional examples of reversible crosslinkers are described inT. W. Green, Protective Groups in Organic Synthesis, John Wiley & Sons(Eds.) (1981). Any variety of strategies used for reversible protectinggroups can be incorporated into a crosslinker suitable for producingcrosslinked protein crystals capable of reversible, controlledsolubilization. Various approaches are listed, in Waldmann's review ofthis subject, in Anaewante Chemie Inl. Ed. Engl., 35, p. 2056 (1996).

[0097] Other types of reversible crosslinkers are disulfidebond-containing crosslinkers. The trigger breaking crosslinks formed bysuch crosslinkers is the addition of reducing agent, such as cysteine,to the environment of the crosslinked protein crystals.

[0098] Disulfide crosslinkers are described in the Pierce Catalog andHandbook (1994-1995).

[0099] Examples of such crosslinkers include:

[0100] Homobifunctional (Symmetric)

[0101] DSS—Dithiobis(succinimidylpropionate), also know as Lomant'sReagent

[0102] DTSSP—3-3′-Dithiobis(sulfosuccinimidylpropionate), water solubleversion of DSP

[0103] DTBP—Dimethyl 3,3′-dithiobispropionimidate.HCl

[0104] BASED—Bis-(β-[4-azidosalicylamido]ethyl)disulfide

[0105] DPDPB—1,4-Di-(3′-[2′-pyridyldithio]-propionamido)butane.

[0106] Heterobifunctional (Asymmetric)

[0107] SPDP—N-Succinimidyl-3-(2-pyridyldithio)propionate

[0108] LC-SPDP—Succinimidyl-6-(3-[2-pyridyldithio]propionate)hexanoate

[0109]Sulfo-LC-SPDP—Sulfosuccinimidyl-6-(3-[2-pyridyldlthio]propionate)hexanoate,water soluble version of LC-SPDP

[0110]APDP—N-(4-[p-azidosalicylamido]butyl)-3′-(2′-pyridyldithio)propionamide

[0111] SADP—N-Succinimidyl(4-azidophenyl)1,3′-dithiopropionate

[0112] Sulfo-SADP—Sulfosuccinimidyl(4-azidophenyl)1,3′-dithiopropionate, water soluble version of SADP

[0113]SAED—Sulfosuccinimidyl-2-(7-azido-4-methycoumarin-3-acetamide)ethyl-1,3′dichiopropionate

[0114]SAND—Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl-1,3′-dithiopropionate

[0115]SASD—Sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate

[0116] SMPB—Succinimidyl-4-(p-maleimidophenyl)butyrate

[0117] Sulfo-SMPB—Sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate

[0118] SMPT—4-Succinimidyloxycarbonyl-methyl-α-(2-pyridylthio)toluene

[0119]Sulfo-LC-SMPT—Sulfosuccinimidyl-6-(α-methyl-α-(2-pyridylthio)toluamido)hexanoate.

[0120] In order that this invention may be better understood, thefollowing examples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any manner.

EXAMPLES Example 1 Preparation of Crosslinked Subtilisin Crystals

[0121] Crystallization of Subtilisin

[0122] One volume of Alcalase 2.5 L (Novo Nordisk Bioindustrials,Franklinton, N.C.) was added to 2 volumes of a solution of 15% sodiumsulfate (pH 5.5) prepared at 30-35° C. The crystallization solution wasseeded with {fraction (1/2,000)}-{fraction (1/500)} volume seeds (30mg/ml slurry of crystals in 15% sodium sulfate (pH 5.5), pH supported at5.5 by adding NaOH. The seeded crystallization solution was incubated at30-35° C., stirring by magnetic stirrer overnight. This yielded 60-80%(by activity) crystal rods, 10-50 μm, in length, 1-3 μm in width, after24-48 hours.

Example 2 Crosslinking of Subtilisin Crystals

[0123] Subtilisin crystals were crosslinked using one of a variety ofcrosslinkers, including: glutaraldehyde, glyoxal, succinaldehyde,octanedialdehyde and epoxides.

[0124] Glutaraldehyde Crosslinking

[0125] Glutaraldehyde (“GA”) (supplied as 50% in aqueous by AldrichChemical Co.) was diluted in deionized water at 4° C. in the variousamounts listed in Table I below. For each ml of subtilisin crystals (27mg/ml) in 15% sodium sulfate, 10 μl of the diluted glutaraldehyde wasadded to the slurry while shaking on a vortex at low speed (for amountsless than 5 ml) or stirring with an overhead stirrer at medium speed(for amounts 25 ml-500 ml). After mixing for the allotted crosslinkingtime, the samples were centrifuged for 20 seconds at maximum speed, thesupernatant was discarded and replaced with 15% sodium sulfate. This“washing” was repeated a total of 5 times. The final resuspension waseffected with 900 μl of 15% sodium sulfate. TABLE I GlutaraldehydeCrosslinking Crosslinking % GA-final GA(ml) H₂O(ml) time (min) 0.00761.0 64.96 60 0.0189 1.0 25.46 39 0.02 1.0 24.0 39, 81 0.05 1.0 9.00 15,60, 89 0.08 1.0 5.25 39, 81 0.10 1.0 4.00 60, 81 0.125 1.0 3.00 3, 10,17, 39 0.15 1.0 2.33 81, 120 0.20 1.0 1.50 19, 60, 120 0.231 1.0 1.1610, 39, 120 0.3 1.0 0.67 60 0.5 1.0 0 60

[0126] Glyoxal Crosslinking

[0127] Glyoxal (supplied as 40% in aqueous by Aldrich Chemical Co.)(“GY”) was added to the crystal suspension to give a final concentrationof 0.01-1.0%. For each ml of subtilisin crystals (27 mg/ml) in 15%sodium sulfate 0.25 μl to 25 ml (0.01 to 1%) of the glyoxal was added tothe slurry, while magnetically stirring at ambient temperature. Afterstirring for 1 hour, the crosslinked crystals were centrifuged andwashed, as described for glutaraldehyde crosslinking.

[0128] Octanedialdehyde Crosslinking

[0129] Octanedialdehyde (“OA”) (100% as supplied by DSM Chemie Linz), inthe amounts shown in Table II below, was added undiluted to 1 ml ofsubtilisin crystal slurry (27 mg/ml in 15% sodium sulfate) whilemagnetically stirring at ambient temperature. Stirring was continued forthe specified time of minutes or hours before the crosslinked crystalswere centrifuged and washed, as described for glutaraldehydecrosslinking. TABLE II Octanedialdehyde Crosslinking % OA - final OA(μl) Crosslinking time 0.05 0.5 16 h 0.1 1.0 16 h 0.2 2.0 16 h 0.25 2.516 h 0.5 5.0 16 h 1.0 10.0 30 m, 1 h, 3 h, 16 h

[0130] Succinaldehyde Crosslinking

[0131] Succinaldehyde (“SA”)(40% as supplied by DSM Chemie Linz) wasadded undiluted, in the amounts shown in Table III below, to 1 ml ofsubtilisin crystal slurry (27 mg/ml in 15% sodium sulfate) whilemagnetically stirring at ambient temperature. Stirring was continued forthe specified time of minutes or hours before the crosslinked crystalswere centrifuged and washed, as described for glutaraldehydecrosslinking. TABLE III Succinaldehyde Crosslinking % SA - final SA (μl)Crosslinking time 1.0 25 30 m, 1 h, 3 h

[0132] Epichlorohydrin Crosslinking

[0133] A 10 μl aliquot of epichlorohydrin (“EP”) (99%, Sigma ChemicalCo., St. Louis, Mo.) was added undiluted to 1 ml of subtilisin crystalslurry (27 mg/ml in 15% sodium sulfate) while stirring at ambienttemperature. Stirring was continued for the specified time of minutes orhours before the crosslinked crystals were centrifuged and washed, asdescribed for glutaraldehyde crosslinking.

[0134] Epoxide Crosslinking

[0135] General Procedure

[0136] Crosslinking of subtilisin was carried out individually using oneof a variety of epoxides. These included:

[0137] 1) General Name—DENACOL

[0138] a) DENACOL EX-411

[0139] b) DENACOL EX-421

[0140] c) DENACOL EX-614

[0141] d) DENACOL EX-201

[0142] e) DENACOL EX-202; all obtained from Nagase American Corporation.

[0143] 2) Obtained from Tokyo Kasei Inc. America:

[0144] a) Neopentyl Glycol diglycidyl Ether (N448) (“NP”)

[0145] b) Ethylene Glycol diglycidyl Ether (EO342)(“EG”).

[0146] The concentration of the epoxide was varied between 0.01 and 4.0%and the crosslinking time was varied from 1 hour to 72 hours. Theprocedure for addition to and removal of crosslinker from enzyme was asdescribed above for glutaraldehyde crosslinking.

[0147] Subsequent crosslinking with glutaraldehyde (0.01 to 0.2%) for (1hour to 5 hours) yielded strongly crosslinked enzyme crystals, insolublein water, but active in the azocasein assay.

[0148] A sample of 1 ml of subtilisin crystal slurry (27 mg/ml in 15%sodium sulfate) was mixed by vortexing at low speed to assure a uniformsuspension of crystals. Epoxide (10% solution in DMF) was added to thecrystal slurry in the amounts specified in Table IV, and the mixture wasshaken at ambient temperature. After the allotted time between 1 and 72hours at ambient temperature, glutaraldehyde (10% in DMF) was added tothe epoxide/crystal mixture and stirring was continued at ambienttemperature for the time specified in Table IV. The resultingcrosslinked enzyme crystals were washed 2× with 1% (NH₄)₂SO₄/10 mM CaCl₂then 3× with water and finally 1× with 1% (NH₄)₂SO₄/10 mM CaCl₂ beforeresuspending in 1% (NH₄)₂SO₄/10M CaCl₂. TABLE IV Epoxide/GlutaraldehydeCrosslinking Epoxide Glutaraldehyde Epoxide Epoxide CrosslinkingGlutaraldehyde Crosslinking Name Amount Time Amount Time EX-411 0.01-4%1-72 h 0.01-0.1% 0.5-2 h EX-421 0.01-4% 1-72 h 0.01-0.1% 0.5-2 h EX-6140.01-4% 1-72 h 0.01-0.1% 0.5-2 h EX-201 0.01-4% 1-72 h 0.01-0.1% 0.5-2 hEX-202 0.01-4% 1-72 h 0.01-0.1% 0.5-2 h NP 0.01-4% 1-72 h 0.01-0.1%0.5-2 h (N448) EG 0.01-4% 1-72 h 0.01-0.1% 0.5-2 h (EO342)

[0149] Large Scale Preparation of A Preferred Epoxide Sample

[0150] Prior to crosslinking, a sample of 380 ml of crystallinesubtilisin in 15% sodium sulfate (27 mg/ml) was mixed by overheadstirring at ambient temperature for 5 minutes to assure a uniformsuspension of crystals. Neopentyl glycol diglycidyl ether (3.838 ml of a10% solution in DMF) was added to the crystal slurry and the mixture wasstirred at ambient temperature. After 5 hours at ambient temperature,3.838 ml of glutaraldehyde (10% in DMF) was added to the epoxide/crystalmixture and stirring was continued at ambient temperature for 1.5 hours.The resulting crosslinked enzyme crystals were washed 2× with 1%(NH₄)₂SO₄/10 mM CaCl₂ then 3× with water and finally 1× with 1%(NH₄)₂SO₄/10 mM CaCl₂, before resuspending in 1% (NH₄)₂SO₄/10 mM CaCl₂′

Example 3 Activity Assay

[0151] In order to test the activity of crosslinked protein crystalsaccording to this invention, as well as other enzyme samples, wedeveloped the following azocasein assay.

[0152] Materials:

[0153] 2.0M Tris Buffer. 500 ppm CaCl₂

[0154] 0.2M Tris Buffer. 50 ppm CaCl₂

[0155] 50% urea

[0156] Azocasein

[0157] 5% trichloroacetic acid (“TCA”)

[0158] Alcalase (2.5 L)

[0159] ChiroCLEC-BL™ (crosslinked subtilisin crystals, available fromAltus Biologics, Inc., Cambridge, Mass.)

[0160] The assay was carried out, preparing azocasein just prior to use,by dissolving 600 mg azocasein with 10 ml of 50% urea and vortexinglightly to complete the dissolution. Then 10 ml 2.0M Tris was added andvortexed to mix, increasing the volume to 100 ml by adding deionizedwater.

[0161] The stock solutions of the enzyme to be assayed in 0.2M Tris wereprepared, to provide 50 μl aliquots to be assayed, as follows:

[0162] Without detergent:

[0163] 0.03 mg/ml Alcalase (soluble, uncrosslinked subtilisin Carlsberg80.3 mg/ml) 3.0 mg/ml ChiroCLEC-BL>.

[0164] With 120 μl detergent/ml solution:

[0165] 0.03 mg/ml Alcalase

[0166] 3.0 mg/ml ChiroCLEC-BL™.

[0167] We added 50 μl aliquots of enzyme to 150 μl of 0.2M Tris andplaced the mixtures in 5 ml test tubes with micro-stir bars. We thenwarmed both the test tubes and the azocasein at 40° C. for 1 minuteusing a metal heating block. After that, we added 1 ml of the azocaseinto each tube and stirred at 40° C. for 15 minutes using the heatingblock at stir speed 4. We then added 2 ml TCA to each tube, mixing byvortex, and placed the tubes in an ice bath immediately, allowing thesamples to stand at 0° C. for 20 minutes. We microfuged the samples for5 minutes at maximum rpm and microfiltered, if necessary. We measuredabsorbance of the expressed activity in abs·units/mg protein·minsupernatant at λ390. In this assay, all measurements were done intriplicate. Controls were void of enzyme but contained detergent if itwas present in the assay. This time=0 assay was repeated at time=15minutes and other times, if necessary.

[0168] The detergents used in the various assays included Tide, Wisk andCiba-Geigy detergents #15, #16 and #44 (“Ciba detergents”). Cibadetergent #15 constitutes a typical European detergentformulation—liquid (aqueous) detergent on the basis of 15% alkylbenzenesulfonate, 14% fatty alcohol ethoxylate and 10% fatty acid salt (soap).Ciba detergent #16 constitutes a typical United States detergentformulation—liquid (aqueous) detergent on the basis of 7.5% alkylbenzene sulfonate, 10% fatty alcohol ethoxylate and 17% alkyl eithersulfate. Ciba detergent #44 constitutes a typical compact detergentformulation—liquid (aqueous) detergent on the basis of 6% fatty alcoholethoxylate, 23% alkyl ether sulfate and 10% sodium citrate. Cibadetergents #15, #16 and #44 may be obtained upon request from CibaSpecialty Chemicals Corp., Division Consumer Care Chemicals, Greensboro,N.C.

[0169] We prepared assay stock solutions from dilution stocks, andcarried out the assays, as follows.

[0170] Activity Assay—200× Dilution—Crosslinked Subtilisin Crystals andCrystalline Subtilisin in Heavy Duty Liquid Detergent (Ciba #15, Ciba#44, Tide and Wisk)

[0171] Stocks A, B, C and D were prepared in 10 ml neoprene tubes asfollows.

[0172] Stock A: Crosslinked Subtilisin Crystals Prepared According tothis Invention (˜27 mg/ml)

[0173] We centrifuged 37 μl slurry of crosslinked subtilisin crystals(equal to 1 mg crosslinked enzyme crystals) to remove supernatant, added1 ml detergent and vortexed to mix. A 50 μl aliquot of the resultingmixture was added to 9.95 ml water, to a final concentration of 5 μg/ml.

[0174] Stock B: Uncrosslinked Subtilisin Crystals (˜27 mg/ml)

[0175] We centrifuged 37 μl slurry of subtilisin crystals (equal to 1 mgenzyme crystals) to remove supernatant, added 1 ml detergent andvortexed to mix. A 50 μl aliquot of the resulting mixture was added to9.95 ml water, to a final concentration of 5 μg/ml.

[0176] Stock C: Alcalase

[0177] We added 18.75 μl Alcalase (80.3 mg/ml) to 3 ml detergent andvortexed to mix. A 50 μl aliquot of the resulting mixture was added to9.95 ml water, to a final concentration of 2.5 μg/ml.

[0178] Stock D: Detergent

[0179] A 50 μl aliquot of detergent was added to 9.95 ml water.

[0180] Azocasein stock (6 mg/ml) was prepared as described above. Upondilution of Stock A and B to 5 μg/ml, the t=0 assay was set upimmediately and carried out as described above, except that the amountof stock sample used was 200 μl, instead of 50 μl+150 μl 0.2M Tris.While the tubes were heating for 1 minute at 40° C., two additionalsamples of 2 ml each of Stocks A, B and C were placed in 1.5 mlmicrocentrifuge tubes and heated to 52° C. while shaking for furthertesting after 5 minutes and 15 minutes dilution with heating.

[0181] Activity Assay—670× Dilution—Crosslinked Subtilisin Crystals andCrystalline Subtilisin in Detergent Concentrate (Ciba #16)

[0182] Stocks A, B, C and D were prepared in 10 ml neoprene tubes asfollows.

[0183] Stock A: Crosslinked Subtilisin Crystals Prepared According tothis Invention (˜27 mg/ml)

[0184] We centrifuged 124 μl slurry of crosslinked subtilisin crystals(equal to 3.35 mg crosslinked enzyme crystals) to remove supernatant,added 1 ml detergent and vortexed to mix. A 50 μl aliquot of theresulting mixture was added to 33.45 ml water, to a final concentrationof 5 μg/ml.

[0185] Stock B: Uncrosslinked Subtilisin Crystals (˜27 mg/ml)

[0186] We centrifuged 124 μl slurry of subtilisin crystals (equal to3.35 mg enzyme crystals) to remove supernatant, added 1 ml detergent andvortexed to mix. A 50 μl aliquot of the resulting mixture was added to33.45 ml water, to a final concentration of 5 μg/ml.

[0187] Stock C: Alcalase

[0188] We added 167 μl Alcalase (80.3 mg/ml) to 8 ml detergent andvortexed to mix. A 50 μl aliquot of the resulting mixture was added to33.45 ml water, to a final concentration of 2.5 μg/ml.

[0189] Stock D: Detergent

[0190] A 50 μl aliquot of detergent was added to 33.45 ml water.

[0191] Azocasein stock (6 mg/ml) was prepared as described above. Thet=0 assay was set up immediately and carried out as described above,except that the amount of stock sample used was 200 μl, instead of 50μl, plus 150 μl 0.2M Tris buffer. While the tubes were heating for 1minute at 40° C., two additional samples of 2 ml each of Stocks A, B andC were placed in Eppendorf tubes and heated to 40° C. while shaking forfurther testing after 5 minutes and 15 minutes dilution with heating.

Example 4 Stability Study

[0192] In order to test the stability of crosslinked enzyme crystalsaccording to this invention, as well as other enzyme samples, wedeveloped the following assays.

[0193] Azocasein Assay—Stability Study 52° C.

[0194] First, we prepared stock solutions of the enzyme samples indetergent in 2 ml Eppendorf tubes with screw caps. After incubating themixtures in a water bath at 52° C. for the appropriate times, we added1.47 ml of 0.2M Tris buffer to one of each enzyme sample tube, and mixedwell. To assay for activity after the appropriate time of incubationfollowed by dilution, we removed a 50 μl aliquot from each tube andassayed as described below. The remaining samples of enzyme/detergentstocks were placed in a water bath at 52° C., with further aliquotsbeing removed for assay at specific times.

[0195] The assay was performed by adding 50 μl enzyme sample to 150 μl0.2M Tris buffer and heating to 40° C. for 1 minute. At a constant 40°C. temperature, we then added 1.0 ml azocasein stock (as described inExample 1) to each sample, stirring for 15 minutes using a heating blockat stir speed 4. We then added 2 ml TCA to each tube, mixing by vortex,and placed the tubes in an ice bath immediately, allowing the samples tostand at 0° C. for 20 minutes. We microfuged the samples for 5 minutesat maximum rpm and microfiltered, if necessary. We measured absorbanceof the supernatant at λ390 and expressed activity as abs·units/mgprotein·min. In this assay, all measurements were done in triplicate.Controls were void of enzyme but contained detergent if it was presentin the assay.

[0196] In order to assess activity as part of stability studies carriedout at 52° C., we prepared assay stock solutions from dilution stocks,and carried out the assays, as follows.

[0197] For Alcalase

[0198] Stocks:

[0199] Stock A: Alcalase (80.3 mg/ml) in commercial detergent (Tide orWisk, deactivated by heating at 70° C. for 4 hours)—finalconcentration=0.25 mg/ml. The stock was prepared by adding 31.2 μlAlcalase to 9.97 ml detergent. A 200 μl aliquot of the resulting mixturewas placed in each of several 2 ml Eppendorf tubes (3× for t=0, 30 andothers).

[0200] Stock B: Alcalase (80.3 mg/ml) in Ciba detergent (Ciba #15, Ciba#16 or Ciba #44)—final concentration=0.25 mg/ml. The stock was preparedby adding 31.2 μl Alcalase to 9.97 ml detergent. A 200 μlaliquot of theresulting mixture was placed in each of several 2 ml Eppendorf tubes (3×for t=0, 1 hour and 4-6 hours).

[0201] Stock C: Commercial detergent (Tide or Wisk, deactivated byheating at 70° C. for 4 hours) 3× (200 μl of the above in 2 ml Eppendorftubes).

[0202] Stock D: Ciba detergent (Ciba #15, Ciba #16 or Ciba #44) 3× (200μl of the above in 2 ml Eppendorf tubes).

[0203] The t=0 assay was performed immediately after 1.47 ml of 0.2 MTris was added to one of each of tubes containing Stocks A-D and thecontents mixed well. Remaining samples of Stocks A-D were placed in awater bath and heated to 52° C. Otherwise, the assays were carried outas described above.

[0204] For Crosslinked Subtilisin Crystals and Crystalline Subtilisin

[0205] Stocks A, B, C and D were prepared in 2 ml Eppendorf tubes withscrew caps as follows.

[0206] Stock A: ChiroCLEC-BL™ in commercial detergent (denatured Tide orWisk)—final concentration=25 mg/ml. The stock was prepared bycentrifuging 3.12 ml enzyme slurry to remove water and then diluting theenzyme to 10 ml with detergent. A 200 μl aliquot of the resultingmixture was placed in each of several 2 ml Eppendorf tubes (4× for t=0,24 hours, 48 hours and 72 hours).

[0207] Stock B: ChiroCLEC-BL™ in Ciba detergent (Ciba #15, Ciba #16 orCiba #44)—final concentration=25 mg/ml. The stock was prepared bycentrifuging 3.12 ml enzyme slurry to remove water and then diluting theenzyme to 10 ml with detergent. A 200 μl aliquot of the resultingmixture was placed in each of several 2 ml Eppendorf tubes (4× for t=0,24 hours, 48 hours and 72 hours at 52° C.).

[0208] Stock C: Commercial detergent (Tide or Wisk, deactivated byheating at 70° C. for 4 hours) 4× (200 μl of the above in 2 ml Eppendorftubes).

[0209] Stock D: Ciba-Geigy detergent (Ciba #15, #16 or #44, depending onwhich detergent was chosen for Stock B) 4× (200 μl of the above in 2 mlEppendorf tubes).

[0210] The t=0 assay was set up immediately after 1.47 ml of 0.2M Triswas added to one of each of tubes containing Stocks A-D and the contentsmixed well. Remaining samples of Stocks A-D were placed in a water bathand heated to 52° C. Otherwise, the assays were carried out as describedabove.

[0211] For Crosslinked Subtilisin Crystals and Crystalline Subtilisin

[0212] Stocks A, B and C were prepared in 2 ml Eppendorf tubes withscrew caps as follows.

[0213] Stock A: Uncrosslinked subtilisin crystals (˜27 mg/ml) in Cibadetergent (Ciba #15, #16 or #44)—final concentration=1 mg/ml. The stockwas prepared by centrifuging 50 μl crystal slurry to remove supernatant,then adding 1.35 ml detergent (Ciba #15, Ciba #16 or Ciba #44) to afinal concentration of 1 mg/ml. An 80 μl aliquot of the resultingmixture was placed in each of several 2.0 ml Eppendorf tubes (3× fort=0, 15 minutes and others).

[0214] Stock B: Crosslinked subtilisin crystals according to thisinvention (˜27 mg/ml) in Ciba detergent (Ciba #15, #16 or #44)—finalconcentration=˜1 mg/ml. The stock was prepared by centrifuging 50 μlcrystal slurry to remove supernatant, then adding 1.35 ml detergent, toa final concentration of 1 mg/ml. An 80 μl aliquot of the resultingmixture was placed in each of several 2.0 ml Eppendorf tubes (3× fort=0, 15 minutes and others).

[0215] Stock C: Ciba detergent (Ciba #15, #16 or #44)-3× (80 μl of theabove in 2.0 ml tubes).

[0216] The t=0 assay was set up immediately after 1.8 ml of water wasadded to one of each of tubes containing Stocks A-C and the contentsmixed well. Remaining samples of Stocks A-C were placed in a water bathand heated to 52° C. Otherwise, the assays were carried out as describedabove, except for the addition of 150 μl 0.2 M Tris buffer instead of200 μl.

[0217] Azocasein Assay—Stability Study 40° C.

[0218] First, we prepared stock solutions of the enzyme samples indetergent in 2 ml Eppendorf tubes with screw caps. To assay stability att=0, we added 1.8 ml of deionized water to a 25 μl of each sample andmixed well. We removed a 25 μl aliquot from each tube and assayed asdescribed below. The remaining samples of enzyme/detergent stocks wereplaced in a water bath at 40° C., with further aliquots being removedfor assay at specific times.

[0219] The assay was performed by adding 25 μl of the diluted enzymesample to 175 μl 0.2M Tris buffer and heating to 40° C. for 1 minute. Ata constant 40° C. temperature, we then added 1.0 ml azocasein stock (asdescribed in Example 0.3) to each sample, stirring for 15 minutes usinga heating block at stir speed 4. We then added 2 ml TCA to each tube,mixing by vortex, and placed the tubes in an ice bath immediately,allowing the samples to stand at 0° C. for 20 minutes. We microfuged thesamples for 5 minutes at maximum rpm and microfiltered, if necessary. Wemeasured absorbance of the supernatant at λ390 and expressed activity asabs·units/mg protein·min. In this assay, all measurements were done intriplicate. Controls were void of enzyme but contained detergent if itwas present in the assay.

[0220] In order to assess activity as part of stability studies carriedout at 40° C., we prepared assay stock solutions from dilution stocks,and carried out the assays, as follows.

[0221] Stock A: Crosslinked Subtilisin Crystals According to thisInvention (˜27 mg/ml)

[0222] We centrifuged 124 μl slurry of crosslinked subtilisin crystals(equal to 3.35 mg crosslinked enzyme crystals) to remove supernatant,added 1 ml detergent and vortexed to mix, to a final concentration of3.35 mg/ml.

[0223] Stock B: Uncrosslinked Subtilisin Crystals (˜27 mg/ml)

[0224] We centrifuged 124 μl slurry of subtilisin crystals (equal to3.35 mg enzyme crystals) to remove supernatant, added 1 ml detergent andvortexed to mix, to a final concentration of 3.35 mg/ml.

[0225] Stock C: Alcalase

[0226] We added 20.9 μl Alcalase (80.3 mg/ml) to 1 ml Ciba detergent(Ciba #15, Ciba #16 or Ciba #44) and commercial detergent (Tide or Wisk,denatured by heating at 70° C. for 4 hours) and vortexed to mix, to afinal concentration of 1.67 mg/ml.

[0227] Stock D: Detergent

[0228] One ml of commercial detergent (Tide or Wisk, deactivated byheating at 70° C. for 4 hours) and Ciba detergents #15, #16 and #44. A25 μl aliquot of each stock was added to 1.8 ml of deionized water andmixed well. A further 25 μl aliquot of the diluted stock was added toeach reaction tube.

[0229] The t=0 assay was performed immediately after 175 μl of 0.2M Triswas added to each tube containing 25 μl of the various stock samples.Remaining samples of Stocks A-D were placed in a water bath and heatedto 0.40° C. Otherwise, the assays were carried out as described above.

Example 5 Dissolution Studies

[0230] We also assessed the characteristics of crosslinked enzymecrystals according to this invention, as well as other enzyme samples,with respect to dissolution in concentrate and upon dilution, asdetailed below. Stock solutions were prepared and diluted as describedabove. The resulting dispersions were heated at 40° C. and analyzedunder a microscope at 250× for dissolution progress.

Example 6 Results of Activity and Stability Assays

[0231] Crosslinked enzyme crystals of subtilisin, as described above, aswell as soluble enzymes and other commercial enzymes, alone and in thepresence of commercial detergents, were tested for activity in theazocasein assay, as described above. Catalyst concentrations forequivalent activities were determined for Alcalase, ChiroCLEC-BL™ Wiskwith active protease and Tide with active protease: ChiroCLEC-BL ™: 150μg/6 μl detergent 0.4-0.5 absorbance units Alcalase: 15 μg/6 μldetergent 0.5-0.6 absorbance units Tide: 6 μl detergent approximately0.6 absorbance units Wisk: 6 μl detergent approximately 0.6 absorbanceunits.

[0232] The dilution studies (discussed supra) were started by assessingthe activities of Alcalase and uncrosslinked crystals of Alcalase inCiba detergents #15 and #16. Initial activities were comparable andlosses of up to −50% were seen after 15 minutes at 52° C.

[0233] Table V summarizes the stability of samples of Alcalase (0.25mg/ml) and ChiroCLEC-BL™ (25 mg/ml) in denatured Wisk or Tide detergent,or in Ciba detergents #15 and #16 at 52° C. Activity was measured by theazocasein assay. TABLE V Stability of Subtilisin in Detergents at 52° C.Alcalase T_(1/2) ChiroCLEC-BL ™ T_(1/2) Detergent at 52° C. at 52° C.#15 less than 15 min >>100 hours #16 less than 15 min >>100 hours Tide(denatured) 16 hours >>100 hours Wisk (denatured) 60-70 hours >>100hours

[0234] We also assessed the stability of various enzymes in Cibadetergent #15 at 52° C. The results are depicted in Table VI below:TABLE VI Stability of Subtilisin in Ciba Detergent #15 at 52° C.Activity 15 min T_(1/2) in Initial dilute concentrate Catalyst Activityat 52° C. 52° C. Alcalase 36 14 ˜15 min Alcalase 33 13 ˜15 min OA 34 18˜15 min 0.1%, 16 h OA 23 17 ˜30 min 1%, 1 h OA 10 14 ˜30 min 1%, 3 h GA27 16 ˜40 min 0.05%, 30 min GA 35 15 ˜40 min 0.05%, 10 min

[0235] All of the crosslinked crystals prepared as described in thetable above which had half-lives in detergent concentrate of ˜30 minutesor more also had good solubility profiles.

[0236] In addition, we assessed the stability of various enzymes in Cibadetergent #15 versus Ciba detergent #16 at 40° C. The results aredepicted in the Table VII below: TABLE VII Stability of Subtilisin inCiba Detergent #15 vs. #16 at 40° C. T_(1/2) in #15 T_(1/2) in #16Initial concentrate concentrate Catalyst Activity at 40° C. at 40° C.Alcalase 33 10 h 2.5 h  GA 27  7 h ˜10 h  0.05%, 30 min OA 32  9 h  8 h0.1%, 16 h OA 15 12 h 16 h 0.2%, 16 h

[0237] We also assessed the effects of crosslinking time on activity andstability of the resulting crosslinked enzyme crystals. These resultsare summarized in the tables below. In Table VIII, an asterisk indicatesvalues measured by incubating 25 μl in a 2 ml tube and “Xs” denotesuncrosslinked protein crystals.

[0238] In preparing the crosslinked protein crystals described in TablesVIII, 1× and X, the protein crystals were crystallized as described inExample 1 and crosslinked with glutaraldehyde as described in Example 2,using the crosslinking times and glutaraldehyde concentrations set forthin that example, or those specified in the tables. TABLE VIIICrosslinking Time vs. Concentration of Glutaraldehyde on Stability ofSubtilisin in Ciba Detergent #16 at 40° C. Activity Activity Stability,Crosslinking abs/mg/min abs/mg/min 18 h GA (%) Time t = 0 t = 18 h % ofXs, t = 0 (Xs) 0 0 33.6 1.1 3.3 (Xs) 0 0 31.7 2.6* 8.2* 0.0189 10.0 28.55.2 16.5 0.0189 10.0 31.3 5.7 17.8 0.0189 39.3 14.1 5.4 17.0 0.0189 39.314.3 5.0 15.8 0.05 5.0 20.7 3.8 12.0 0.05 15.0 16.4 7.0 22.1 0.05 18.619.6 8.7 27.4 0.05 18.6 17.8 9.8 30.9 0.05 60.0 0 13.5 42.6 0.05 60.03.0 14.7 46.4 0.125 3.0 18.3 9.1 28.7 0.125 3.0 15.4 9.0 28.4 0.125 10.07.9 14.9 46.9 0.125 10.0 9.5 14.8 46.6 0.125 10.0 7.9 14.7 46.4 0.12510.0 9.5 13.4 42.1 0.125 10.0 9.4 12.2 38.5 0.125 10.0 8.1 12.2 38.50.125 10.0 8.3 16.0 50.5 0.125 17.0 5.4 14.0 44.2 0.125 17.0 6.4 15.348.3 0.125 39.3 2.1 3.3 10.4 0.125 39.3 1.0 5.8 18.3 0.125 39.3 1.1 4.413.9 0.125 39.3 1.7 4.5 14.3 0.125 39.3 1.6 5.7 18.0 0.125 39.3 0.9 3.310.4 0.125 68.6 1.3 3.1 9.9 0.2 5.0 10.4 12.1 38.2 0.2 15.0 2.5 9.0 28.40.2 18.6 1.8 6.9 21.8 0.2 18.6 0.8 3.1 9.8 0.2 60.0 0.4 1.3 4.1 0.2 60.01.4 1.4 4.4 0.231 10.0 2.7 13.0 41.0 0.231 10.0 4.8 11.7 37.0 0.231 39.30.5 1.1 3.5 Alcalase 28.0 3.1* 9.8* Alcalase 32.9 0.2 1.0

[0239] In Table IX, an asterisk indicates that crystals were crushedduring crosslinking and dash marks indicate that no measurements weretaken at those points. All samples were prepared at 1 ml (27 mg) scale.TABLE IX Activity of Glutaraldehyde Crosslinked Subtilisin in CibaDetergent #16 at 40° C. Cross- linking Activity at 40° C. time(abs/mg/min) GA (%) (min) t = 0 18 h 39 h 63 h 80 h 90 h 6 days (Xs) 0 033.6  1.1 — — — — — 0.0076 60 14.1  3.0 — — — — — 0.02 39 12.0  7.7 — —— — — 0.02 80 6.9 11.5 — — — 0   — 0.02* 80 12.2 26.2 10.7  3   — — —0.05* 31 13.5 26.3 9.8 5.3 — — — 0.05 60 4.9 17.7 7.9 2.4 — — 1.0 0.05*60 8.7 27.3 12.5  7.3 — — — 0.05 89 1.3 12.1 — — — 3.7 — 0.08 39 2.212.6 — — — 5.3 — 0.08 81 0.8  3.9 — — 5.8 — 3.9 0.08* 81 9.8 — — — 10.2 — — 0.125 3 16.4  9.7 1.7 0.3 — — — 0.125 10 9.4 14.8 9.4 1.9 — — —0.125 17 6.4 15.3 11.5  8.5 4.6 — — 0.2 5 10.4 12.7 5.1 0.3 — — — 0.2310 4.8 11.7 10.0  8.5 5.2 — — Alcalase 30.5  1.6

[0240] TABLE X Conditions for Larger Scale Crosslinked Enzyme CrystalPreparation - Stability of Glutaraldehyde Crosslinked Subtilisin in CibaDetergent #16 at 40° C. Crosslinking time Stability at 40° C. GA (%)(min) (abs/mg/min) t = 0 18 h (Xs) 0 33.9 — t = 0 16 h 38 h 59 h 110 h*0.05 60 23.2  16.8 5.9 1.9 5.6 *0.08 80 14.4  12.2 4.1 2.4 — *0.1 808.0 13.6 5.5 3.1 1.4 *0.125 60 9.3 20.9 13.6  — 2.3 *0.15 80 3.8 11.27.4 5.2 2.8 *0.231 60 5.2  9.9 9.3 6.6 8.3 *1.0 60 1.3  2.5 2.2 1.1 2.4t = 0 24 h 48 h 72 h 120 h 168 h 264 h § 0.25 120 4.8 9.5 7.7 6.7 5.74.3 4.4 § 0.20 120 2.6 9.5 8.6 8.7 5.6 — 4.5 § 0.15 120 5.2 14.3  9.65.6 3.7 — 1.2 § 0.1-NP/ 5 h/1.5 h 9.6 10.2  6.3 — — 5.7  0.1 GA

[0241] In Table X, an asterisk indicates that crosslinkings were carriedout at a 1-2 g scale, § indicates that crosslinkings were carried out ata 10 g scale on previously crushed crystals and dash marks indicate thatno measurements were taken at those points.

[0242]FIG. 1 graphically depicts the stability of 10 g scalepreparations of crosslinked subtilisin crystals according to thisinvention in Ciba detergent #16 at 40° C. In FIG. 1, “Altus I”represents crystals crosslinked with 0.25% glutaraldehyde for 2 hours;“Altus II” represents crystals crosslinked with 0.20% glutaraldehyde for2 hours; “Altus III” represents crystals crosslinked with 0.15%glutaraldehyde for 2 hours and “Altus IV” represents crystalscrosslinked with 0.1% neopentyl glycol diglycidyl ether for 5 hours,followed by 0.1% glutaraldehyde for 1.5 hours. All the crosslinkedsamples were crushed prior to crosslinking using a Brinkman PolytronHomogenizer, then prepared on a 10 g scale and monitored by theazocasein assay over one week at 40° C.

Example 7 Results of Dissolution Study

[0243] The dissolution study demonstrated whether various crosslinkedenzyme crystals dissolve in concentrate and the extent to which theydissolve upon dilution under conditions of use, for example under washconditions. Representative results of this test are included in thetables below, in which “+” indicates that the sample dissolved, “−”indicates that the sample did not dissolve, “−/+” indicates that thesample dissolved somewhat (1 mg/ml in detergent liquid). In the tables,“GP” denotes crystals crosslinked as described infra for GA crosslinkingusing, instead, ultrapure glutaraldehyde (supplied as an 8% aqueoussolution by the Sigma Chemical Co.) which was not diluted prior toaddition to the protein crystals. TABLE XI Detergent Liquid IncubationStudy - Dissolution Study - Concentrate 14 h at 40° C. Catalyst Ciba #15Ciba #16 Ciba #44 Tide OA − −/+ + − 1%, 16 h OA − − + − 0.5%, 16 h OA− + + − 0.1%, 16 h OA − −/+ + − 0.2%, 16 h GA − −/+ + − 0.5%, 1 h GA − −− − 0.9%, 1h GA − − + − 0.7%, 1 h EP −/+ + + − 1.0%, 20 min GP − −/+ + −0.08%, 20 min CLECBL ™ − − − − Crystals + + + − (uncross- linked)

[0244] TABLE XII Detergent Liquid Incubation Study - Dissolution Study -200 fold Dilution 20 minutes at 52° C. Catalyst Ciba #15 Ciba #16 Ciba#44 Tide OA −/+ −/+ + − 1%, 16 h OA − + + − 0.5%, 16 h OA − + + + 0.1%,16 h OA −/+ + + − 0.2%, 16 h GA − −/+ + − 0.5%, 1 h GA − + + − 0.9%, 1 hGA − − + − 0.7%, 1 h EP + + + + 1.0%, 20 min GP − + + − 0.08%, 20 minCLECBL ™ − − − − Crystals + + + + (uncross- linked)

[0245] As demonstrated in the tables above, crosslinked enzyme crystalsaccording to this invention are essentially insoluble in concentrateddetergent and essentially soluble in diluted detergent under washconditions.

Example 8 Summary of Properties of Crosslinked Enzyme Crystals of thisInvention

[0246] Table XIII below summarizes the overall stability/instability,activity and dissolution properties in Ciba detergent #15 of crosslinkedsubtilisin crystals prepared according to this invention usingdialdehydes. TABLE XIII Cross- Solubility Solubility Activity Stabilitylinker in Ciba #15 on Dilution (t = 0) at 52° C. Glyoxal low dissolve athigh low 52° C. Succini- low dissolve at 17-66% of ND maldehyde 52° C.;Alcalase partially dissolve at 25° C. Glutaral- very low dissolve at1-100% of low 52° C. dehyde 52° C.; Alcalase moderate partially to 40°C. fully dissolve at 25° C. Octane- very low % dissolve at 30-66% of low52° C. dialdehyde 52° C.; Alcalase moderate partially to 40° C. fullydissolve at 25° C.

[0247] As demonstrated in Table XIII above, the crosslinked enzymecrystals of the present invention are insoluble and, therefore, stableunder storage conditions, while quickly releasing their activity underconditions of use. Advantageously, the crosslinked enzyme crystals ofthis invention exhibit activity similar to their soluble oruncrosslinked crystallized counterparts under conditions of use, whiledisplaying 5-6 fold improved stability, as well as favorable dissolutionproperties.

Example 9 Effect of Change of Chemical Composition on CrosslinkedSubtilisin Crystals

[0248] We crystallized subtilisin as described in Example 1 andcrosslinked the resulting crystals as described in Example 2, using GA1%/1 hour. When 100 μL (2.2 mg) of the resulting crosslinked subtilisincrystals was suspended in 1.5 mL of 33.3% of acetonitrile/phosphatebuffer (0.3 M, pH 7.5), the crystals were completely dissolved after 45minutes at 40° C.

[0249] Using similar conditions, suspending the crosslinked subtilisincrystals in 1.2 mL of 16.7% acetonitrile/buffer, the crystals werecompletely dissolved after 5 hours. Activity (U) in 100% ACN/16.7%ACN/33.3% time buffer Buffer Buffer 0 27.3 27.3 27.3 1.5 h 27.3 —  7.4*3.3 h 27.3 25.5 7.0 h 27.3  24.0**

[0250] Assay: 0.2 mmol (75.8 mg) of TAME in 2.5 mL phosphate buffer wasincubated with each crosslinked subtilisin crystal sample (equal to0.044 mg enzyme crystals) suspension (or solution) at room temperature.One unit hydrolyzed 1.0 mmole of TAME per min. from per mg crosslinkedcrystals.

[0251] The results above illustrate the trigger of addition of organicsolvent to the environment of crosslinked protein crystals of thisinvention.

Example 10 Wash Performance of Detergents Containing CrosslinkedSubtilisin Crystals

[0252] We assessed the activity and storage stability of crosslinkedenzyme crystals of this invention in liquid detergent, using a washingassay designed to test the ability of the detergent to remove stainsfrom a fabric.

Washing Assay

[0253] Preparation of Fabric

[0254] Cloth samples of the same size and weight were cut from the samebolt:

[0255] 5 g of soiled test cloth and

[0256] 5 g of cotton ballast with no soil (Ciba No. 1-3005).

[0257] Prior to washing the samples, we measured the light intensity(=lightness) remitted by the soiled fabric samples (as described below).

[0258] Preparation of Detergent Solution

[0259] The sample of liquid detergent to be tested was heated in a flaskfor two hours at 20° C. The sample was then homogenized by vigorousshaking and 0.8 g of the detergent was removed from the flask and addedto 200 ml of tap water (20° C.) in a metallic beaker. The aqueousdetergent solution was stirred for 60 seconds.

[0260] Washing

[0261] A sample of soiled test cloth and a sample of unsoiled ballastwere placed together into the beaker containing the aqueous detergentsolution. The beaker was closed tightly and immediately inserted into apre-heated (40° C.) washing machine (Unitest, manufactured by Hereus,Switzerland). During the washing process, the beaker was rotatedconstantly in a water bath heated to 40° C. As a result, the contents ofthe beaker continuously warmed, up to a temperature of 40° C.

[0262] Exactly 20 minutes after the fabric was placed in the detergentsolution, washing was stopped and the washed fabric was immediatelyremoved from the detergent solution and rinsed for 30 seconds with coldtap water (13-15° C.). The wet fabric was centrifuged and ironed toremove wrinkles and dried at the same time.

[0263] Measurement of Washing Performance

[0264] Each sample of the washed and dried fabric was examined for stainremoval by remission measurements (lightness Y) between 460 and 700 nmusing a Spectraflash 500 (Datacolor). A cut off filter was used toeliminate potential interference by contamination with UV-absorbingmaterials. The lightness value of each test cloth was measured 5× and anaverage calculated.

[0265] With increasing washing performance, the lightness of the fabricincreases Washing performance is thus defined as a difference inlightness, ΔY:

ΔY=Lightness of fabric after washing−Lightness of fabric before washing

Example 11 Effect of Concentration of Crosslinked Subtilisin Crystals onWashing Performance of Detergents Containing them

[0266] Washing performance of crosslinked enzyme crystals according tothis invention was examined as a function of their concentration in theliquid detergent, using the materials described below.

[0267] Test fabric: EMPA (Eidgen{overscore (o)}ssischeMaterialpr{overscore (u)}fungs und Forschungsanstalt, St. Gallen,Switzerland) #116 soiled with a combination of blood, milk and carbonblack.

[0268] Liquid detergent: Ciba detergent #16.

[0269] Enzyme:

[0270] Crosslinked enzyme crystals; sample Altus IV (as described inExample 6)

[0271] Uncrosslinked enzyme (Alcalase).

[0272] Concentration of Enzyme in Liquid Detergent:

[0273] enzyme concentrations were between 0.05 and 0.9 w % (dry matterweight). Table XIV provides further details. TABLE XIV Weight of enzymeDry matter Liquid suspension (g) weight of Detergent Enzyme w % AlcalaseAltus IV enzyme g Ciba #16 g 0.05 0.106 0.0053 10 0.05 0.130 0.0056 100.1 0.207 0.0104 10 0.1 0.240 0.0104 10 0.3 0.599 0.0301 10 0.3 0.6830.0297 10 0.5 0.492 0.0247 5 0.5 0.580 0.0252 5 0.9 0.886 0.0445 5 0.91.046 0.0455 5

[0274] Preparation of Liquid Detergent with Enzyme

[0275] Specific aliquots of the suspension of enzyme crystals (see TableXIV) were added to a flask and centrifuged to separate the crystals fromthe liquid. The liquid was discarded and the crystals were suspended andhomogenized in the liquid detergent (for quantities see Table XIV). Theresulting preparations were used in the washing tests.

[0276] Washing tests to evaluate the performance of the enzyme detergentformulations were carried out as described in the assay above. Theresults of the study are depicted in FIG. 2. The figure demonstratesthat at enzyme concentrations ≧0.1 w %, the washing effect of Cibaliquid detergent #16 formulated with Altus IV exceeds that of theformulation with uncrosslinked Alcalase. The efficacy of bothcrosslinked and uncrosslinked enzymes was reduced at enzymeconcentrations below 0.1 w %.

Example 12 Storage Stability and Washing Performance of DetergentsContaining Crosslinked Subtilisin Crystals

[0277] Detergents formulated with crosslinked and uncrosslinked enzymeswere stored at a constant temperature, in order to examine enzymestability in concentrated liquid detergent. The detergent formulations(150 g each) were prepared by the same procedure as the samples inExample 10.

[0278] Liquid detergent: Ciba detergent #16.

[0279] Enzyme:

[0280] Crosslinked enzyme crystals: sample Altus IV (Example 6)

[0281] Uncrosslinked enzyme (Alcalase) Enzyme concentration: 0.3 w %(dry matter) in liquid detergent.

[0282] Storage Temperature for Stability Studies:

[0283] All samples were stored at 30° C. for between 0 and 7 days. After7 days, the samples were divided after 7 days into two equal portions,in order to study stability at elevated temperature. One portioncontinued to be stored at 30° C., while the other was stored at 40° C.

[0284] Test fabric: Three different soiled fabrics were used. All ofthem were standard test materials available from EMPA:

[0285] EMPA #112: cocoa soiled fabric

[0286] EMPA #116: blood, milk and carbon black soiled fabric

[0287] EMPA #111: blood soiled fabric.

[0288] Washing Performance on Cocoa Soiled Fabric

[0289] Washing performance of various enzyme formulated liquiddetergents was studied with respect to removal of cocoa stains from acocoa soiled test fabric, using the washing assay described above.Storage stability was determined by assessing washing performanceperiodically during the detergent storage time, thus monitoring theimpact of storage temperature on enzyme performance in the liquiddetergent. In this assay, the effectiveness of the liquid detergentdecreases as enzyme stability degrades. The results of this assay, shownin FIGS. 3 and 4, are discussed below.

[0290] Storage Stability at 30° C.

[0291] As demonstrated in FIG. 3, both Alcalase and Altus IV formulateddetergents exhibited an improved performance after 2 days of storage(compared to initial values). However, as storage time increased, theperformance of the Alcalase formulation decreased continuously overtime, while the Altus IV formulated detergent exhibited no degradation,even after 28 days of storage.

[0292] Storage Stability at 40° C.

[0293] As demonstrated in FIG. 4, when the temperature was raised from30 to 40° C., Alcalase formulated detergent lost activity within 2 days,while the Altus IV formulated detergent degraded slightly, whileremoving the cocoa soil from the test fabric significantly, even after21 days of storage at 40° C.

[0294] Washing Performance on Fabric Soiled by a Combination of Blood,Milk and Carbon Black (EMPA #116 Test Fabric)

[0295] The experimental conditions and detergents were the same (exceptthe stained fabric) as for washing of cocoa stains. The results of thewashing tests are depicted in FIGS. 5 and 6.

[0296] Storage Stability at 30° C.

[0297]FIG. 5 clearly illustrates the decay of washing performance of theAlcalase formulated detergent after 2 days of storage at 30° C. However,liquid detergent containing Altus IV enzyme maintained its originalwashing performance, even after 28 days of storage.

[0298] Storage Stability at 40° C.

[0299] As demonstrated in FIG. 6, when the storage temperature wasraised from 30 to 40° C., Alcalase formulated detergent lost nearly allof its washing performance within 2 days. In contrast, detergentcontaining Altus IV retained its washing power for an additional 14days.

[0300] Washing Performance on Fabric Soiled with Blood

[0301] Washing performance on blood stains was tested with enzymecontaining detergents stored at 30° C. The detergent composition,washing conditions were the same as in washing of cocoa stains. Theresults of the washing test are illustrated in FIG. 7.

[0302] The assays show that the washing effect on blood stain by Ciba#16 liquid detergent formulated with Alcalase was low in comparison todetergent without enzyme. On the other hand, the Altus IV formulationwas more active in washing conditions and more stable in storage.

[0303] Storage Stability at 30° C.

[0304] The washing effect of Alcalase formulated detergent decreasedrapidly with storage time, whereas Altus IV formulated detergentretained almost completely its full capacity after 28 days of storage.

Example 13 Solubility of Crosslinked Subtilisin Crystals at 30° C. and37° C.

[0305] We studied the solubility of various subtilisin crystals, whichhad been crosslinked with glutaraldehyde (GA), octanedialdehyde (OA),neopentyl glycol diglycidyl ether (NP) followed by glutaraldehyde, orDENACOL EX-411 (411) followed by glutaraldehyde.

[0306] In 1.5 ml Eppendorf tubes, samples of uncrosslinked subtilisincrystals and crosslinked subtilisin crystal slurry, equal to 37.5 mg ofenzyme, were microfuged at 5,000 rpm for 5 min and the supernatantliquid was removed. A 1.5 ml aliquot of PBS buffer (0.01 M phosphate,0.0027 M potassium chloride, 0.137 M sodium chloride, pH 7.4) was addedto each sample, bringing the concentration of subtilisin to 25 mg/ml.The samples were transferred to 2 ml glass vials with screw caps andmagnetic stir bars then were incubated at 30° C. or at 37° C. Sampleswere studied for dissolution by periodically removing 50 μl of theslurry, microfuging at 13,000 rpm for 5 mins, removing 20 μl of thealiquot and placing it in 980 μl of deionized water, then measuring UVabsorbance at 280 nm.

[0307] The following samples were studied: Crosslinker CrosslinkerConcentration Crosslinking Time GA  1.0% 1.5 h  GA 0.25%  2 h GA  0.2% 2 h GA 0.15%  2 h NP/GA 0.1%/0.1%   5 h/1.5 h 411/GA 0.015%/0.035% 16h/1 h OA  0.2% 16 h OA  0.1% 16 h OA 0.05% 16 h

[0308] The solubility profiles of the samples, shown in FIGS. 8 and 9,illustrate different rates of dissolution for the crosslinked crystals.

Example 14 Reversible Crosslinkers—Disulfide Crosslinked SubtilisinCrystals

[0309] We prepared subtilisin crystals (30-40 μm average, 27 mg/ml inNa₂SO₄) as previously described for subtilisin crystallization.

[0310] We then crosslinked the crystals using one of the followingcrosslinkers:

[0311] 1) Dimethyl 3,3′-dithiobispropionimidate.HCl—(DTBP) (Pierce)

[0312] 2) Dithiobis(succinimidylpropionate)—(DSP)(Pierce)

[0313] 3) 3,3′-Dithiobis (sulfosuccinimidylpropionate)—(DTSSP) (Pierce).

[0314] Crosslinking was carried out in 15 ml neoprene screw cap tubes byplacing 740 μl of subtilisin crystal slurry (20 mg) in 9.26 ml of buffer(25 mM NaCO₃/50 mM NaHCO₃, pH 8.0). One crosslinker was added to eachtube as follows: A) 93 mg DTBP (30 mM) B) 100 mg DTSSP (16 mM) C) 120 mgDSP (30 mM).

[0315] The tubes were tumbled at ambient temperature (24-26° C.) untilall samples were determined to be insoluble in 32 mM NaOH (5 days)-100μl sample in 300 μl NaOH. Uncrosslinked samples were readily soluble in32 mM NaOH at the same concentrations. Crosslinking was stopped by theaddition of 1 ml of 1 M Tris, pH 7.5. The samples were centrifuged at3,000 rpm for 5 minutes, the supernatant removed and replaced by 5 ml of100 mM Tris, pH 7.5. Centrifugation at 3,000 rpm, for 5 min, followed byreplacement of supernatant with 5 ml of 100 mM Tris (pH 7.5) wasrepeated 3×.

Example 15 Dissolution of Disulfide Bond-Containing CrosslinkedSubtilisin Crystals

[0316] A 200 mM solution of cysteine was prepared by dissolving 121 mgcysteine in 5 ml 100 mM Tris (pH 7.5). A 400 μl aliquot of the cysteinesolution was added to 3×750 μl vials. A 400 μl aliquot of 100 mM Tris(pH 7.5) was added to another 3×750 μl vials. Each crosslinked sample(100 μl) was added to one vial containing cysteine and one vial withoutcysteine. All samples were incubated at 37° C. and monitored fordissolution of crosslinked enzyme crystals (direct visual andmicroscopic observation).

[0317] After incubation for 3 hrs at 37° C., the DTBP sample appeared tobe fully soluble in the presence of cysteine and insoluble in itsabsence. The DTSSP sample appeared to be nearly fully soluble in thepresence of cysteine and insoluble in its absence. The DSP sample wasbarely soluble in the presence of cysteine and insoluble in its absence.

Example 16 Crystallization of Candida Rugosa Lipase

[0318] A 5 kg aliquot of Candida rugosa lipase (“CRL”) in powder form(Meito) was mixed with 5 kg celite and dissolved in 102 L distilleddeionized water and the volume brought to 200 L with the deionizedwater. The suspension was mixed in an Air Drive Lightning Mixer for 2hours at room temperature and then filtered through a 0.5 micron filterto remove celite. The mixture was then ultrafiltered and concentrated to14 L (469 g) using a 3K hollow fiber filter membrane cartridge. Solidcalcium acetate was added to a concentration of 5 mM Ca(CH₃COO)₂. The pHwas adjusted to pH 5.5 with concentrated acetic acid as necessary. A 7litre aliquot was crystallized by either addition of 1.75 litres of2-methyl-2,4-pentanediol (“MPD”) or by addition of 3.5 litres of a 30%solution of PEG-8000. The resulting solution was mixed andcrystallization allowed to proceed overnight at ambient temperature forabout 17-20 hrs. The crystal yield was about 70%.

[0319] Recrystallization

[0320] The Candida rugosa lipase crystals were solubilized by theaddition of 50 mM sodium phosphate (pH 5.2). Soluble proteinconcentration of the crystallization solution was adjusted to 20 mg/ml.MPD was added gradually with stirring over a 6-hour period, to a finalconcentration of 25%. The resulting solution was mixed andcrystallization allowed to proceed at ambient temperature for 20 hours.

Example 17 Crystallization of Candida Rugosa Lipase

[0321]Candida rugosa lipase crystals prepared as described in Example16, prior to the solubilization and recrystallization steps, weresolubilized by the addition of 50 mM sodium acetate (pH 6.5). Solubleprotein concentration of the crystallization solution was adjusted to 20mg/ml. MPD was added gradually with stirring over a 6-hour period to afinal concentration of 20%. The resulting solution was mixed andcrystallization allowed to proceed at ambient temperature for 20 hours.

Example 18 Crosslinking of Candida Rugosa Lipase Crystals

[0322]Candida rugosa lipase crystals, prepared as described in Example16, were crosslinked by addition of untreated neat glutaraldehyde(Sigma) by adding 2 ml of 20% glutaraldehyde stepwise in a 40.5 mlvolume over one hour to 8 ml of stirred lipase crystals (25 mg/ml), atambient temperature. The final crosslinker concentration was-4.0%.Crosslinking was allowed to proceed over 24 hours. Crystals wererecovered by low speed centrifugation and washed with 25% MPD in 50 mMsodium phosphate (pH 5.2).

Example 19 Crosslinking of Candida Rugosa Lipase Crystals

[0323]Candida rugosa lipase crystals, prepared as described in Example16, were crosslinked by addition of untreated neat glutaraldehyde byadding 2 ml of 20% glutaraldehyde gradually over a one hour period.Crystals were crosslinked and processed as described in Example 18.

Example 20 Crosslinking of Candida Rugosa Lipase Crystals

[0324]Candida rugosa lipase crystals, prepared as described in Example16, were crosslinked as described in in Example 19, except that thereaction was allowed to proceed for 24 hours. The crystals were thenprocessed as described in Example 18.

Example 21 Crosslinking of Candida Rugosa Lipase Crystals

[0325]Candida rugosa lipase crystals, prepared as described in Example17, were crosslinked by addition of glutaraldehyde to a finalconcentration of 4.0%. Crosslinking was allowed to proceed for 3 hours.The crystals were processed as described in Example 18.

Example 22 Crosslinking of Candida Rugosa Lipase Crystals

[0326]Candida rugosa lipase crystals, prepared as described in Example17, were crosslinked in neat glutaraldehyde at a concentration of 6.5%for 1 hour. Crosslinking and processing were performed as described inExample 18.

Example 23 Crosslinking of Candida Rugosa Lipase Crystals

[0327]Candida rugosa lipase crystals, prepared as described in Example17, were crosslinked in neat glutaraldehyde at a concentration of 6.0%for 1 hour. Crosslinking and processing were performed as described inExample 18.

Example 24 pH Controlled Solubility of Crosslinked Candida Rugosa LipaseCrystals

[0328] Solubility of various crosslinked Candida rugosa lipase crystalswas studied following an increase in pH from 6.5 to 9.0. The crystalswere incubated at 1 mg/ml in 50 mM sodium phosphate (pH 9) containing25% MPD. Aliquots were removed after 3 hour and 24 hour incubation at25° C. with stirring. Activity and soluble protein concentration weremeasured as described in Example 25. The results are described in thetable below. Time (hr) Crosslinked 3 24 Crystal [Prot.] [Prot.]Preparation Activity (U) (mg/ml) Activity (U) (mg/ml) Example 18 7.50.47 20 1 Example 19 10.8 0.60 11.3 0.63 Example 20 7.5 0.42 8.8 0.49

Example 25 pH Solubility of Crosslinked Candida Rugosa Lipase Crystals

[0329] Solubility of various crosslinked Candida rugosa lipase crystalswas studied following an increase in pH from 5.2 to 7.5. The crystalswere incubated at 1 mg/ml in 50 mM sodium phosphate (pH 7.5) containing25% MPD. Aliquots were removed after 3 hour and 24 hour incubation at25° C. with stirring. Insoluble material was removed by filtration (0.25micron). Activity in solution was measured spectrophotometrically bymonitoring the hydrolysis of para nitrophenyl acetate (Fluka) at 400 nm.Substrate concentration was 1 mM. The assay was performed at 25° C. in a1 ml volume of 50 mM sodium acetate (pH 6.5). Soluble proteinconcentration was measured by absorbance at 280 mm. Results arepresented in the table below. Time (hr) Crosslinked 3 24 Crystal [Prot.][Prot.] Preparation Activity (U) (mg/ml) Activity (U) (mg/ml) Example 212.4 0.12 15 0.91 Example 22 10.0 0.63 15 1.0 Example 23 2.5 0.17 11 0.69

Example 26 Crystallization of Human Serum Albumin

[0330] Human serum albumin (“HSA”) was purchased from Sigma ChemicalCompany as a lyophilized powder. We added 10 grams of protein powder toa 75 ml stirred solution of 100 mM phosphate buffer pH 5.5 at 4° C.Final protein concentration was 120 mg/ml (determined from OD₂₈₀,extinction coefficient for serum albumin was assumed to equal 1).Saturated ammonium sulfate solution (767 g/l) prepared in deionizedwater was added to the protein solution to a final concentration of 50%saturation (350 g/l). The crystallization solution was “seeded” with 1ml of albumin crystals (50 mg/ml) in 50% ammonium sulfate (pH 5.5). Seedcrystals were prepared by washing a sample of crystals free ofprecipitate with a solution of 50% saturated ammonium sulfate in 100 mMphosphate buffer (pH 5.5). The seeded crystallization solution wasincubated at 4° C. overnight on a vigorously rotating platform. Crystalrods (20μ) appeared in the solution overnight (16 hr).

Example 27 Crosslinking of Human Serum Albumin Crystals

[0331] We crosslinked human serum albumin crystals, prepared asdescribed in Example 18, at 4° C. in a 10 ml stirred solution ofcrystals and mother liquor containing 50% saturated ammonium sulfate, asdescribed above. The crystals, which were not washed prior tocrosslinking, were crosslinked with glutaraldehyde as supplied by themanufacturer (Sigma). Glutaraldehyde (“GA”) (20%) was added to thestirred crystallization solution in. 4 equal volumes (62.5 μl) at 15minute intervals to a final concentration of 0.5% (250 μl GA). Thecrystals were then incubated at 4° C. Aliquots were removed atincubation times 0, 30 min, 60 min and 4 hours incubation. Crosslinkedalbumin crystals were collected by low speed centrifugation and washedrepeatedly with pH 7.5, 100 mM Tris HCl, 4° C. Washing was stopped whenthe crystals could be centrifuged at high speed without aggregation.

Example 28 Crosslinking of Human Serum Albumin Crystals

[0332] We crosslinked human serum albumin crystals as described inExample 27 above, with one modification; glutaraldehyde (20%) was addedto the crystallization solution in 4 equal volumes (131.3 μl) at 15minute intervals to a final concentration of 1% (525 μl GA).

Example 29 Solubility of Human Serum Albumin Crystals Crosslinked in0.5% GA, Time: 0 Minutes Incubation. Dissolution Induced by ElevatedTemperature

[0333] Human serum albumin, crystallized as described in Example 26 andcrosslinked for 0 minutes in 0.5% glutaraldehyde, as described inExample 27, was assayed for solubility by incubating the crystals (20mg/ml) with stirring, in phosphate buffered saline solution (pH 7.5) atroom temperature (“RT”) or at 37° C. Aliquots were removed for assay attimes 0.5, 1, 4 and 24 hours. Insoluble material was removed from thesolution by centrifugation and the soluble protein concentration wasmeasured spectrophotometrically at 280 nm, as indicated in Table XV.TABLE XV Soluble Protein (mg/ml) Time (hr) RT 37° C. 0.5 0.3 1.5 1.0 3 54.0 4 12.5 24.0 17 18.5

Example 30 Solubility of Human Serum Albumin Crystals Crosslinked in0.5% GA, Time: 30 Minutes Incubation. Dissolution Induced by ElevatedTemperature

[0334] Human serum albumin, crystallized as described in Example 26 andcrosslinked in 0.5% glutaraldehyde, as described in Example 27, wasassayed for solubility by incubating the crystals (20 mg/ml) inphosphate buffered saline solution (pH 7.5) at room temperature or at37° C. Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours.Insoluble material was removed from the solution by centrifugation andthe soluble protein concentration was measured spectrophotometrically at280 nm, as indicated in Table XVI. TABLE XVI Soluble Protein (mg/ml)Time (hr) RT 37° C. 0.5 1.5 4 1.0 3 5.5 4.0 7 10 24.0 13.5 17.5

Example 31 Solubility of Human Serum Albumin Crystals Crosslinked in0.5% GA, Time: 60 Minutes Incubation. Dissolution Induced by ElevatedTemperature

[0335] Human serum albumin, crystallized as described in Example 26 andcrosslinked with 0.5% glutaraldehyde, as described in Example 27, wasassayed for solubility by incubating the crystals (20 mg/ml) inphosphate buffered saline solution (pH 7.5) at room temperature or at37° C. Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours.Insoluble material was removed from the solution by centrifugation andthe soluble protein concentration was measured spectrophotometrically at280 nm, as indicated in Table XVII. TABLE XVII Soluble Protein (mg/ml)Time (hr) RT 37° C. 0.5 0 0.4 1.0 0 0.6 4.0 0 3 24.0 8 17

Example 32 Solubility of Human Serum Albumin Crystals Crosslinked in0.5% GA, Time: 240 Minutes Incubation. Dissolution Induced by ElevatedTemperature

[0336] Human serum albumin, crystallized as described in Example 26 andcrosslinked with 0.5% glutaraldehyde, as described in Example 27, wasassayed for solubility by incubating the crystals (20 mg/ml) inphosphate buffered saline solution (pH 7.5) at room temperature or at37° C. Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours.Insoluble material was removed from the solution by centrifugation andthe soluble protein concentration was measured spectrophotometrically at280 nm, as indicated in Table XVIII. TABLE XVIII Soluble Protein (mg/ml)Time (hr) RT 37° C. 0.5 0 0 1.0 0.5 0 4.0 3.5 3 24.0 8.5 14.5

Example 33 Solubility of Human Serum Albumin Crystals Crosslinked in1.0% GA, Time: 0 Minutes Incubation. Dissolution Induced by ElevatedTemperature

[0337] Human serum albumin, crystallized as described in Example 26 andcrosslinked as described in Example 27, was assayed for solubility byincubating the crystals (20 mg/ml) in phosphate buffered saline solution(pH 7.5) at room temperature or at 37° C. Aliquots were removed forassay at times 0.5, 1, 4 and 24 hours. Insoluble material was removedfrom the solution by centrifugation and the soluble proteinconcentration was measured spectrophotometrically at 280 nm, asindicated in Table XIX. TABLE XIX Soluble Protein (mg/ml) Time (hr) RT37° C. 0.5 1 2 1.0 3 7 4.0 10.5 16 24.0 19 18.5

Example 34 Solubility of Human Serum Albumin Crystals Crosslinked in1.0% GA, Time: 30 Minutes Incubation. Dissolution Induced by ElevatedTemperature

[0338] Human serum albumin, crystallized as described in Example 26 andcrosslinked as described in Example 27, was assayed for solubility byincubating the crystals (20 mg/ml) in phosphate buffered saline solution(pH 7.5) at room temperature or at 37° C. Aliquots were removed forassay at times 0.5, 1, 4 and 24 hours. Insoluble material was removedfrom the solution by centrifugation and the soluble proteinconcentration was measured spectrophotometrically at 280 nm, asindicated in Table XX. TABLE XX Soluble Protein (mg/ml) Time (hr) RT 37°C. 0.5 0 0 1.0 0 2 4.0 4.5 7 24.0 8 13

Example 35 Solubility of Human Serum Albumin Crystals Crosslinked in1.0% GA Time: 60 Minutes Incubation. Dissolution Induced by ElevatedTemperature

[0339] Human serum albumin, crystallized as described in Example 26 andcrosslinked as described in Example 27, was assayed for solubility byincubating the crystals (20 mg/ml) in phosphate buffered saline solution(pH 7.5) at room temperature or at 37° C. Aliquots were removed forassay at times 0.5, 1, 4 and 24 hours. Insoluble material was removedfrom the solution by centrifugation and the soluble proteinconcentration was measured spectrophotometrically at 280 nm, asindicated in Table XXI. TABLE XXI Soluble Protein (mg/ml) Time (hr) RT37° C. 0.5 0 0.5 1.0 0 1.5 4.0 1 4 24.0 9 13.5

Example 35 Solubility of Human Serum Albumin Crystals Crosslinked in1.0% GA, Time: 240 Minutes Incubation. Dissolution Induced by ElevatedTemperature

[0340] Human serum albumin, crystallized as described in Example 26 andcrosslinked as described in Example 27, was assayed for solubility byincubating the crystals (20 mg/ml) in phosphate buffered saline solution(pH 7.5) at room temperature or at 37° C. Aliquots were removed forassay at times 0.5, 1, 4 and 24 hours. Insoluble material was removedfrom the solution by centrifugation and the soluble proteinconcentration was measured spectrophotometrically at 280 nm, asindicated in Table XXII. TABLE XXII Soluble Protein (mg/ml) Time (hr) RT37° C. 0.5 0 0 1.0 0 0 4.0 0 2 24.0 6 10.3

Example 36 Crystallization of Thermolysin

[0341] Thermolysin was purchased from Diawa (Japan) as a lyophilizedpowder. Fifteen grams of protein powder were added to a 100 ml stirredsolution of 10 mM calcium acetate (pH 11) at ambient temperature. The pHwas maintained at 11 by addition of 2 N NaOH, until the thermolysin wascompletely solubilized. The pH was then adjusted to pH 7.5 by additionof 2 N acetic acid. Crystallization was allowed to proceed overnight at4° C. Final protein concentration was 40 mg/ml (determined from OD₂₈₀,extinction coefficient for thermolysin was assumed to equal 1.8).Crystals were recovered by centrifugation and recrystallized to obtain amore uniform crystal size. Recrystallization was performed in a mannernearly identical to that described for the initial crystallization.Crystals (40 mg/ml protein) were dissolved by addition of base at roomtemperature. The pH of the crystallization solution was adjusted to 6.5and crystallization was permitted to proceed at ambient temperature.Crystal rods (50μ) appeared in the solution overnight (16 hr).

Example 37 Crosslinking of Thermolysin Crystals

[0342] Thermolysin crystals, prepared as described in Example 36, weresuspended (50 mg/ml) in a 50 mM solution of sodium acetate (pH 6.5).Crystals were crosslinked with glutaraldehyde as supplied by themanufacturer (Sigma). Ten milliliters of glutaraldehyde (10%) were addedgradually over a 1 hour period with stirring to a 10 ml suspension ofcrystals. After all of the glutaraldehyde was added, the crystallizationsolution incubated at ambient temperature. Aliquots were removed atincubation times 0.5, 1 and 3 hr. Crosslinked crystals were collected bylow speed centrifugation and washed exhaustively with pH 7.5 50 mM TrisHCl, containing 10 mM calcium acetate.

Example 38 Solubility of Thermolysin Crystals Crosslinked for 0.5 hr.Dissolution Induced by Removal of Calcium Ions by EDTA

[0343] Thermolysin, crystallized as described in Example 36 andcrosslinked for 0.5 hr as described in Example 37, was assayed forsolubility by incubating the crystals (1 mg/ml) with stirring, in 10 mMTris HCl (pH 7.2) containing 1 mM EDTA (Sigma) 40° C. One ml aliquotswere removed for assay at times 0.5, 3 and 24 hours. Insoluble crystalswere removed from the solution by filtration. One ml of 500 mM calciumacetate (pH 7.2) was added to each aliquot. Soluble proteinconcentration was measured spectrophotometrically at 280 nm. Enzymaticactivity was measured spectrophotometrically by monitoring thehydrolysis of a dipeptide substrate, FAGLA (Feder). Substrateconcentration was 1.67 mM. One unit is defined as the amount of enzymerequired to hydrolyze 1 mmole of substrate in one minute at pH 7.2, 40°C. The activity of soluble thermolysin was 27 U/mg protein. Data ispresented in Table XXIII. TABLE XXIII Soluble Protein Activity Time (hr)(% of Max) (% of Max) 0.5 80 30 3.0 103 97 24.0 100 91

Example 39 Solubility of Thermolysin Crystals Crosslinked for 1 hr.Dissolution Induced by Removal of Calcium Ions by EDTA

[0344] Thermolysin, crystallized as described in Example 36 andcrosslinked for 1 hr as described in Example 37, was assayed forsolubility by incubating the crystals (1 mg/ml) with stirring, in 10 mMTris HCl (pH 7.2) containing 1 mM EDTA 40° C. One ml aliquots wereremoved for assay at times 0.5, 3 and 24 hours. Insoluble crystals wereremoved from the solution by filtration. One ml of 500 mM calciumacetate (pH 7.2) was added to each aliquot. Soluble proteinconcentration was measured spectrophotometrically at 280 nm. Enzymaticactivity was measured spectrophotometrically by monitoring thehydrolysis of a dipeptide substrate, FAGLA (Feder). Substrateconcentration was 1.67 mM. One unit is defined as the amount of enzymerequired to hydrolyze 1 μmole of substrate in one minute at pH 7.2, 40°C. The activity of soluble thermolysin was 27 U/mg protein. Data ispresented in Table XXIV. TABLE XXIV Soluble Protein Activity Time (hr)(% of Max) (% of Max) 0.5 7 11 3.0 24 29 24.0 104 87

Example 40 Solubility of Thermolysin Crystals Crosslinked for 3 hr.Dissolution Induced by Removal of Calcium Ions by EDTA

[0345] Thermolysin, crystallized as described in Example 36 andcrosslinked for 3 hr as described in Example 37, was assayed forsolubility by incubating the crystals (1 mg/ml) with stirring, in 10 mMTris HCl (pH 7.2) containing 1 mM EDTA 40° C. One ml aliquots wereremoved for assay at times 0.5, 3 and 24 hours. Insoluble crystals wereremoved from the solution by filtration. One ml of 500 mM calciumacetate (pH 7.2) was added to each aliquot. Soluble proteinconcentration was measured spectrophotometrically at 280 nm. Enzymaticactivity was measured spectrophotometrically by monitoring thehydrolysis of a dipeptide substrate, FAGLA (Feder). Substrateconcentration was 1.67 mM. One unit is defined as the amount of enzymerequired to hydrolyze 1 μmole of substrate in one minute at pH 7.2, 40°C. The activity of soluble thermolysin was 27 U/mg protein. Data ispresented in Table XXV. TABLE XXV Soluble Protein Activity Time (hr) (%of Max) (% of Max) 0.5 2 0 3.0 2 0 24.0 100 73

Example 41 Solubility of Thermolysin Crystals Crosslinked for 3 hr.Dissolution Induced by Removal of Calcium Ions by Dilution

[0346] Thermolysin crystals, prepared as described in Example 36 andcrosslinked for 3 hr as described in Example 37, were washed free ofcalcium containing buffer and assayed for solubility by incubating thecrystals (1 mg/ml) with stirring in deionized water. One ml aliquotswere removed for assay at times 0.5, 3 and 24 hours. Insoluble crystalswere removed from the solution by filtration. One ml of 500 mM calciumacetate (pH 7.2) was added to each aliquot. Soluble proteinconcentration was measured spectrophotometrically at 280 nm. Enzymaticactivity was measured spectrophotometrically by monitoring thehydrolysis of a dipeptide substrate, FAGLA (Feder). Substrateconcentration was 1.67 mM. One unit is defined as the amount of enzymerequired to hydrolyze 1 μmole of substrate in one minute at (pH 7.2),40° C. The activity of soluble thermolysin was 27 U/mg protein. Data ispresented in Table XXVI. TABLE XXVI Soluble Protein Activity Time (hr)(% of Max) (% of Max) 0.5 0 0 3.0 0 7 24.0 111 81

Example 42 Crystallization of Glucose Isomerase

[0347] Glucose isomerase (“GA”) was supplied by Cultor (Finland) as acrystal slurry. The enzyme was recrystallized by solubilizing a 50 mlvolume of the crystal slurry at 50° C. with stirring for 15 minutes. Thesolution was clarified by filtration and allowed to cool slowly at roomtemperature. Fifty micron crystals appeared within 5 hours. Crystalswere recovered by low speed centrifugation and washed with 166 mMmagnesium sulfate.

Example 43 Crosslinking of Glucose Isomerase Crystals

[0348] Five hundred milligrams of glucose isomerase crystals, preparedas described in Example 42, were suspended in a 50 ml solution of 166 mMmagnesium sulfate. The crystals were crosslinked with glutaraldehyde assupplied by the manufacturer (Sigma). Five milliliters of glutaraldehyde(10%) were added gradually over a 1 hour period with stirring to the 50ml suspension. After all of the glutaraldehyde was added, thecrystallization solution incubated at ambient temperature. Aliquots wereremoved at incubation times 1, 3 and 24 hr. Crosslinked crystals werecollected by low speed centrifugation and washed exhaustively with 50 mMTris HCl (pH 7.0).

Example 44 Solubility of Glucose Isomerase Crystals Crosslinked for 1hr. Dissolution Induced by Removal of Calcium Ions by Dilution at 50° C.

[0349] Glucose isomerase crystals, prepared as described in Example 42and crosslinked for 1 hr as described in Example 43, assayed forsolubility by incubating the crystals (1 mg/ml) with stirring indeionized water. One ml aliquots were removed for assay at times 1, 3and 24 hours. Soluble protein concentration was measuredspectrophotometrically at 280 nm (OD280) (extinction coefficient for GIwas assumed to equal 1). Enzymatic activity was measuredspectrophotometrically by monitoring the conversion of fructose toglucose.

[0350] Glucose concentration was quantitated spectrophotometricallyusing a coupled enzyme assay containing hexokinase andglucose-6-phosphate dehydrogenase. The dehydrogenase uses NADP as acofactor and the amount of NADPH formed in the reaction isstoichiometric with the concentration of substrate (glucose). The assaywas purchased as a kit from Boehringer Mannheim and was used accordingto the manufacturer's instructions. One unit is defined as the amount ofenzyme required to convert 1 μmole fructose to glucose in one minute atpH 7.0, 60° C. The activity of soluble glucose isomerase was 51 U/mgprotein. Data is presented in Table XXVII. TABLE XXVII Soluble ProteinActivity Time (hr) (% of Max) (% of Max) 0.5 8 0 3.0 52 31 24.0 100 57

Example 45 Solubility of Glucose Isomerase Crystals Crosslinked for 3hr. Dissolution Induced by Removal of Calcium Ions by Dilution at 50° C.

[0351] Glucose isomerase crystals, prepared as described in Example 42and crosslinked for 3 hr as described in Example 43, were assayed forsolubility by incubating the crystals (1 mg/ml) with stirring indeionized water. One ml aliquots were removed for assay at times 1, 3and 24 hours. Soluble protein concentration was measuredspectrophotometrically at 280 nm (OD280) (extinction coefficient for GIwas assumed to equal 1). Enzymatic activity was measuredspectrophotometrically by monitoring the conversion of fructose toglucose.

[0352] Glucose concentration was quantitated spectrophotometricallyusing the coupled enzyme assay containing hexokinase andglucose-6-phosphate dehydrogenase, as described in Example 44. Data ispresented in Table XXVIII. TABLE XXVIII Soluble Protein Activity Time(hr) (% of Max) (% of Max) 0.5 2 0 3.0 10 6.5 24.0 86 43

Example 46 Solubility of Glucose Isomerase Crystals Crosslinked for 24hr. Dissolution Induced by Removal of Calcium Ions by Dilution at 50° C.

[0353] Glucose isomerase crystals, prepared as described in Example 42and crosslinked for 1 hr as described in Example 43, were assayed forsolubility by incubating the crystals (1 mg/ml) with stirring indeionized water. One ml aliquots were removed for assay at times 1, 3and 24 hours. Soluble protein concentration was measuredspectrophotometrically at 280 nm (OD280) (extinction coefficient forglucose isomerase was assumed to equal 1). Enzymatic activity wasmeasured spectrophotometrically by monitoring the conversion of fructoseto glucose.

[0354] Glucose concentration was quantitated spectrophotometricallyusing the coupled enzyme assay containing hexokinase andglucose-6-phosphate dehydrogenase, as described in Example 44. Data ispresented in Table XXIX. TABLE XXIX Soluble Protein Activity Time (hr)(% of Max) (% of Max) 0.5 2 0 3.0 24 5 24.0 83 61

Example 47 Preparation of Tablets Containing Crosslinked ProteinCrystals According to this Invention

[0355] Tablets containing crosslinked protein crystals according to thisinvention may be prepared as follows. A suspension of crosslinkedprotein crystals is placed in 0.1 M sodium acetate, 20 mM calciumchloride and buffer (pH 7) and dried at 35° C. The resulting driedmaterial may be mixed with sorbitol 50:50 by weight and granulated withEudragit NE 30D (a neutral copolymer based on ethyl- and methylacrylate)or Eudagit RL 30D (an ammonio-methacrylate copolymer). The granules aredried (for example, for 16 hours at 40° C.) and compressed to roundtablets of about 5 mm diameter and weight of about 125 mg. The contentof crosslinked protein crystals in such solid preparations is about 45%by weight. If the above-described preparation is made without usingsorbitol, the resulting tablets contain about 63% by weight crosslinkedprotein crystals.

[0356] When introduced into water or aqueous buffer (such as theabove-described acetate buffer) all the tablets disintegrate in a matterof 10 minutes under mild shaking at room temperature) producingparticles less than 100 μm in size, the majority in the range of 4-10μm. Microscopic examination reveals polymer-free singular proteincrystals, as the predominant species. The slurry obtained by disruptingthe tablets is assayed titrimetrically using hydrolysis of N(α)-p-tosylL-arginine methyl ester (TAME) at 25° C. (pH 8). Activity correspondingto between about 50% and 80% of activity of an equal amount ofcrosslinked protein crystals (counting the indicated weight of thecrosslinked crystals, rather than of the whole tablets) results.

[0357] While we have hereinbefore described a number of embodiments ofthis invention, it is apparent that our basic constructions can bealtered to provide other embodiments which utilize the processes andcompositions of this invention. Therefore, it will be appreciated thatthe scope of this invention is to be defined by the claims appendedhereto rather than by the specific embodiments which have been presentedhereinbefore by way of example.

1. A crosslinked protein crystal, said protein crystal being capable ofchange from insoluble and stable form to soluble and active form upon achange in the environment surrounding said crystal, said change beingselected from the group consisting of: change in temperature, change inpH, change in chemical composition, change from concentrate to diluteform, change in shear force acting upon the crystal and combinationsthereof.
 2. The crosslinked protein crystal according to claim 1,wherein said change from concentrate to dilute form comprises a changein solute concentration.
 3. The crosslinked protein crystal according toclaim 2, wherein said change in solute concentration comprises anincrease or decrease in salt concentration.
 4. The crosslinked proteincrystal according to claim 3, wherein said change in soluteconcentration comprises a decrease in salt concentration.
 5. Thecrosslinked protein crystal according to claim 2, wherein said change insolute concentration comprises an increase or decrease in waterconcentration.
 6. The crosslinked protein crystal according to claim 5,wherein said change in solute concentration comprises an increase inwater concentration.
 7. The crosslinked protein crystal according toclaim 2, wherein said change in solute concentration comprises anincrease or decrease in organic solvent concentration.
 8. Thecrosslinked protein crystal according to claim 2, wherein said change insolute concentration comprises a decrease in detergent concentration. 9.The crosslinked protein crystal according to claim 2, wherein saidchange in solute concentration comprises a decrease in proteinconcentration.
 10. The crosslinked protein crystal according to claim 1,wherein said change from concentrate to dilute form comprises a changein concentration of all solutes from about 2-fold to about 10,000-fold.11. The crosslinked protein crystal according to claim 10, wherein saidchange from concentrate to dilute form comprises a change inconcentration of all solutes from about 2-fold to about 700-fold. 12.The crosslinked protein crystal according to claim 1, wherein saidchange in pH comprises a change from acidic pH to basic pH.
 13. Thecrosslinked protein crystal according to claim 1, wherein said change inpH comprises a change from basic pH to acidic pH.
 14. The crosslinkedprotein crystal according to claim 1, wherein said change in temperaturecomprises an increase or decrease in temperature.
 15. The crosslinkedprotein crystal according to claim 14, wherein said change intemperature is an increase in temperature from a low temperature betweenabout 0° C. and about 20° C. to a high temperature between about 25° C.and about 70° C.
 16. The crosslinked protein crystal according to claim1, wherein said active form of said protein is a form which is activeagainst macromolecular substrates.
 17. A crosslinked protein crystal,said protein crystal having a half-life of activity under storageconditions which is greater than at least 2 times that of the solubleform of the protein that is crystallized to form said crystal that iscrosslinked and activity similar to that of the soluble form of theprotein under conditions of use.
 18. A crosslinked protein crystal, saidprotein crystal being capable of releasing its protein activity at acontrolled rate upon exposure to a change in the environment surroundingsaid crystal, said change being selected from the group consisting ofchange in pH, change in solute concentration, change in temperature,change in chemical composition, change in shear force acting upon thecrystals and combinations thereof.
 19. The crosslinked protein crystalaccording to claim 18, wherein said controlled rate of releasing proteinactivity is determined by a factor selected from the group consistingof: the degree of crosslinking of said crosslinked protein crystal, thelength of time of exposure of protein crystal to the crosslinker, therate of addition of the crosslinking agent to said protein crystal, thenature of the crosslinker, the chain length of the crosslinker, thesurface area of said crosslinked protein crystal, the size of saidcrosslinked protein crystal, the shape of said crosslinked proteincrystal and combinations thereof.
 20. The crosslinked protein crystalaccording to claim 18, wherein said crystal has a protein activityrelease rate of between about 0.1% per day and about 100% per day. 21.The crosslinked protein crystal according to claim 18, wherein saidcrystal has a protein activity release rate between about 0.01% per hourand about 100% per hour.
 22. The crosslinked protein crystal accordingto claim 18, wherein said crystal has a protein activity release ratebetween about 1% per minute and about 50% per minute.
 23. Thecrosslinked protein crystal according to any one of claims 1, 17 or 18,said protein crystal being substantially insoluble and stable in acomposition under storage conditions and substantially soluble andactive under conditions of use of said composition.
 24. The crosslinkedprotein crystal according to claim 23, wherein said composition isselected from the group consisting of cleaning agents, detergents,personal care compositions, cosmetics, pharmaceuticals, veterinarycompounds, vaccines, foods, feeds, diagnostics and formulations fordecontamination.
 25. The crosslinked protein crystal according to claim24, wherein said detergent is selected from the group consisting ofpowdered detergents, liquid detergents, bleaches, household cleaners,hard surface cleaners, industrial cleaners, carpet shampoos andupholstery shampoos.
 26. The crosslinked protein crystal according toclaim 24, wherein said cosmetic is selected from the group consisting ofcreams, emulsions, lotions, foams, washes, gels, compacts, mousses,sunscreens, slurries, powders, sprays, foams, pastes, ointments, salves,balms, shampoos, sunscreens and drops.
 27. The crosslinked proteincrystal according to any one of claims 1, 17 or 18, wherein said proteinis an enzyme.
 28. The crosslinked protein crystal according to claim 27,wherein said enzyme is selected from the group consisting of hydrolases,isomerases, lyases, ligases, transferases and oxidoreductases.
 29. Thecrosslinked protein crystal according to claim 28, wherein said enzymeis selected from the group consisting of proteases, amylases,cellulases, lipases and oxidases.
 30. The crosslinked protein crystalaccording to any one of claims 1, 17 or 18, wherein said protein isselected from the group consisting of therapeutic proteins, cleaningagent proteins, personal care proteins, veterinary proteins, foodproteins, feed proteins, diagnostic proteins and decontaminationproteins.
 31. The crosslinked protein crystal according to any one ofclaims 1, 17 or 18, wherein said protein is selected from the groupconsisting of hormones, antibodies, inhibitors, growth factors, trophicfactors, cytokines, lymphokines, toxoids, growth hormones, nerve growthhormones, bone morphogenic proteins, and nutrients.
 32. The crosslinkedprotein crystal according to any one of claims 1, 17 or 18, wherein saidprotein is selected from the group consisting of insulin, amylin,erythropoietin, Factor VIII, TPA, dornase-α, α-1-antitrypsin, urease,fertility hormones, FSH, LSH, postridical hormones, tetanus toxoid anddiptheria toxoid.
 33. The crosslinked protein crystal according to anyone of claims 1, 17 or 18, said crystal having a longest dimension ofbetween about 0.01 μm and about 500 μm.
 34. The crosslinked proteincrystal according to any one of claims 1, 20 or 21, said crystal havinga longest dimension of between about 0.1 μm and about 50 μm.
 35. Thecrosslinked protein crystal according to any one of claims 1, 17 or 18,said crystal having a shape selected from the group consisting of:spheres, needles, rods, plates, rhomboids, cubes, bipyramids and prisms.36. A composition comprising a crosslinked protein crystal according toany one of claims 1, 17 or 18, said composition being selected from thegroup consisting of cleaning agents, detergents, personal carecompositions, cosmetics, pharmaceuticals, veterinary compounds,vaccines, foods, feeds, diagnostics and formulations fordecontamination.
 37. The composition according to claim 36, wherein saiddetergent is selected from the group consisting of powdered detergents,liquid detergents, bleaches, household cleaners, hard surface cleaners,industrial cleaners, carpet shampoos and upholstery shampoos.
 38. Thecomposition according to claim 36, wherein said cosmetic is selectedfrom the group consisting of creams, emulsions, lotions, foams, washes,gels, compacts, sunscreens, slurries, powders, sprays, foams, pastes,ointments, salves, balms, shampoos, sunscreens and drops.
 39. A proteindelivery system, said system comprising crosslinked protein crystalsaccording to any one of claims 1, 17 or
 18. 40. The protein deliverysystem according to claim 39, wherein said protein is selected from thegroup consisting of: detergent enzymes, cosmetic proteins,pharmaceutical proteins, agricultural proteins, vaccine proteins anddecontamination proteins.
 41. The protein delivery system according toclaim 40, said protein delivery system being a microparticulate proteindelivery system.
 42. The protein delivery system according to claim 41,wherein said microparticulate protein delivery system comprisescrosslinked protein crystals having a longest dimension between about0.01 μm and about 500 μm.
 43. The protein delivery system according toclaim 42, wherein said microparticulate protein delivery systemcomprises crosslinked protein crystals having a longest dimension ofbetween about 0.1 μm and about 50 μm.
 44. The protein delivery systemaccording to claim 41, wherein said microparticulate protein deliverysystem comprises crosslinked protein crystals having a shape selectedfrom the group consisting of: spheres, needles, rods, plates, rhomboids,cubes, bipyramids and prisms.
 45. A detergent formulation comprising acrosslinked protein crystal according to any one of claims 1, 17 or 18.46. A controlled release formulation comprising a crosslinked proteincrystal according to any one of claims 1, 17 or
 18. 47. A pharmaceuticalcontrolled release formulation comprising a crosslinked protein crystalaccording to any one of claims 1, 17 or
 18. 48. A pharmaceuticalcontrolled release formulation comprising a crosslinked protein crystal,said crystal being substantially insoluble under storage conditions andcapable of releasing its protein activity in vivo at a controlled rate.49. The pharmaceutical controlled release formulation according to claim47, said pharmaceutical being capable of administration by parenteral ornon-parenteral routes.
 50. The pharmaceutical controlled releaseformulation according to claim 49, said pharmaceutical being capable ofadministration by oral, pulmonary, nasal, aural, anal, dermal, ocular,intravenous, intramuscular, intraarterial, intraperitoneal, mucosal,sublingual, subcutaneous or intracranial route.
 51. The pharmaceuticalcontrolled release formulation according to claim 47, wherein saidpharmaceutical is capable of administration by oral route and saidcrosslinked protein crystal is substantially insoluble under gastric pHconditions and substantially soluble under small intestine pHconditions.
 52. A vaccine comprising a crosslinked protein crystalaccording to any one of claims 1, 17 or
 18. 53. A formulation comprisinga crosslinked protein crystal according to any one of claims 1, 17 or18, said formulation being selected from the group consisting oftablets, liposomes, granules, spheres, microspheres, microparticles andcapsules.
 54. A method for producing crosslinked protein crystalscomprising the step of reacting protein crystals with a firstcrosslinking agent, or a first crosslinking agent and at least a secondcrosslinking agent, under conditions sufficient to induce crosslinkingof said crystals to the extent that the resulting crosslinked crystalsare characterized by the ability to change from insoluble and stableform to soluble and active form upon a change in their environment, saidchange being selected from the group consisting of change intemperature, change in pH, change in chemical composition, change fromconcentrate to dilute form, change in shear force acting upon thecrystals and combinations thereof.
 55. A method for producingcrosslinked protein crystals comprising the step of reacting proteincrystals with a first crosslinking agent, or a first crosslinking agentand at least a second crosslinking agent, under conditions sufficient toinduce crosslinking of said crystals to the extent that the resultingcrosslinked crystals are characterized by a half-life of activity understorage conditions which is greater than at least 2 times that of thesoluble form of the protein that is crystallized to form said crystalsthat are crosslinked and activity similar to that of the soluble form ofthe protein under conditions of use.
 56. A method for producingcrosslinked protein crystals comprising the step of reacting proteincrystals with a first crosslinking agent, or a first crosslinking agentand at least a second crosslinking agent, under conditions sufficient toinduce crosslinking of said crystals to the extent that the resultingcrosslinked crystals are characterized by being capable of releasingtheir protein activity at a controlled rate upon exposure to a change intheir environment, said change being selected from the group consistingof change in pH, change in soluble concentration, change in temperature,change in chemical composition, change in shear force acting upon thecrystals and combinations thereof. 57-85 (canceled)